CRISPR-Cas and CRISPR-based screening system for precise gene editing and targeted cancer therapy.

Target cancer therapy has been developed for clinical cancer treatment based on the discovery of CRISPR (clustered regularly interspaced short palindromic repeat) -Cas system. This forefront and cutting-edge scientific technique improves the cancer research into molecular level and is currently widely utilized in genetic investigation and clinical precision cancer therapy. In this review, we summarized the genetic modification by CRISPR/Cas and CRISPR screening system, discussed key components for successful CRISPR screening, including Cas enzymes, guide RNA (gRNA) libraries, target cells or organs. Furthermore, we focused on the application for CAR-T cell therapy, drug target, drug screening, or drug selection in both ex vivo and in vivo with CRISPR screening system. In addition, we elucidated the advantages and potential obstacles of CRISPR system in precision clinical medicine and described the prospects for future genetic therapy.In summary, we provide a comprehensive and practical perspective on the development of CRISPR/Cas and CRISPR screening system for the treatment of cancer defects, aiming to further improve the precision and accuracy for clinical treatment and individualized gene therapy.

Enhancing antibacterial properties by regulating valence configurations of copper: a focus on Cu-carboxyl chelates.

Optimizing the antibacterial effectiveness of copper ions while reducing environmental and cellular toxicity is essential for public health. A copper chelate, named PAI-Cu, is skillfully created using a specially designed carboxyl copolymer (a combination of acrylic and itaconic acids) with copper ions. PAI-Cu demonstrates a broad-spectrum antibacterial capability both in vitro and in vivo, without causing obvious cytotoxic effects. When compared to free copper ions, PAI-Cu displays markedly enhanced antibacterial potency, being about 35 times more effective against Escherichia coli and 16 times more effective against Staphylococcus aureus. Moreover, Gaussian and ab initio molecular dynamics (AIMD) analyses reveal that Cu+ ions can remain stable in the carboxyl compound's aqueous environment. Thus, the superior antibacterial performance of PAI-Cu largely stems from its modulation of copper ions between mono- and divalent states within the Cu-carboxyl chelates, especially via the carboxyl ligand. This modulation leads to the generation of reactive oxygen species (˙OH), which is pivotal in bacterial eradication. This research offers a cost-effective strategy for amplifying the antibacterial properties of Cu ions, paving new paths for utilizing copper ions in advanced antibacterial applications.

Protein Allostery Study in Cells Using NMR Spectroscopy.

Protein allostery is commonly observed in vitro. But how protein allostery behaves in cells is unknown. In this work, a protein monomer-dimer equilibrium system was built with the allosteric effect on the binding characterized using NMR spectroscopy through mutations away from the dimer interface. A chemical shift linear fitting method was developed that enabled us to accurately determine the dissociation constant. A total of 28 allosteric mutations were prepared and grouped to negative allosteric, nonallosteric, and positive allosteric modulators. ∼ 50% of mutations displayed the allosteric-state changes when moving from a buffered solution into cells. For example, there were no positive allosteric modulators in the buffered solution but eight in cells. The change in protein allostery is correlated with the interactions between the protein and the cellular environment. These interactions presumably drive the surrounding macromolecules in cells to transiently bind to the monomer and dimer mutational sites and change the free energies of the two species differently which generate new allosteric effects. These surrounding macromolecules create a new protein allostery pathway that is only present in cells.

Cytoplasmatic Localization of Six1 in Male Testis and Spermatogonial Stem Cells.

Sine oculis homeobox 1 (Six1) is an important factor for embryonic development and carcinoma malignancy. However, the localization of Six1 varies due to protein size and cell types in different organs. In this study, we focus on the expression and localization of Six1 in male reproductive organ via bioinformatics analysis and immunofluorescent detection. The potential interacted proteins with Six1 were also predicted by protein-protein interactions (PPIs) and Enrichr analysis. Bioinformatic data from The Cancer Genome Atlas and Genotype-Tissue Expression project databases showed that SIX1 was highly expressed in normal human testis, but low expressed in the testicular germ cell tumor sample. Human Protein Atlas examination verified that SIX1 level was higher in normal than that in cancer samples. The sub-localization of SIX1 in different reproductive tissues varies but specifically in the cytoplasm and membrane in testicular cells. In mouse cells, single cell RNA-sequencing data analysis indicated that Six1 expression level was higher in mouse spermatogonial stem cells (mSSCs) and differentiating spermatogonial than in other somatic cells. Immunofluorescence staining showed the cytoplasmic localization of Six1 in mouse testis and mSSCs. Further PPIs and Enrichr examination showed the potential interaction of Six1 with bone morphogenetic protein 4 (Bmp4) and catenin Beta-1 (CtnnB1) and stem cell signal pathways. Cytoplasmic localization of Six1 in male testis and mSSCs was probably associated with stem cell related proteins Bmp4 and CtnnB1 for stem cell development.

Genetic evidence for differential functions of figla and nobox in zebrafish ovarian differentiation and folliculogenesis.

FIGLA and NOBOX are important oocyte-specific transcription factors. Both figla-/- and nobox-/- mutants showed all-male phenotype in zebrafish due to increased dominance of the male-promoting pathway. The early diversion towards males in these mutants has precluded analysis of their roles in folliculogenesis. In this study, we attenuated the male-promoting pathway by deleting dmrt1, a key male-promoting gene, in figla-/- and nobox-/- fish, which allows a sufficient display of defects in folliculogenesis. Germ cells in figla-/-;dmrt1-/- double mutant remained in cysts without forming follicles. In contrast, follicles could form well but exhibited deficient growth in nobox-/-;dmrt1-/- double mutants. Follicles in nobox-/-;dmrt1-/- ovary could progress to previtellogenic (PV) stage but failed to enter vitellogenic growth. Such arrest at PV stage suggested a possible deficiency in estrogen signaling. This was supported by lines of evidence in nobox-/-;dmrt1-/-, including reduced expression of ovarian aromatase (cyp19a1a) and level of serum estradiol (E2), regressed genital papilla (female secondary sex characteristics), and more importantly the resumption of vitellogenic growth by E2 treatment. Expression analysis suggested Nobox might regulate cyp19a1a by controlling Gdf9 and/or Bmp15. Our discoveries indicate that Figla is essential for ovarian differentiation and follicle formation whereas Nobox is important for driving subsequent follicle development.

Engineering the Active Site Lid Dynamics to Improve the Catalytic Efficiency of Yeast Cytosine Deaminase.

Conformational dynamics is important for enzyme catalysis. However, engineering dynamics to achieve a higher catalytic efficiency is still challenging. In this work, we develop a new strategy to improve the activity of yeast cytosine deaminase (yCD) by engineering its conformational dynamics. Specifically, we increase the dynamics of the yCD C-terminal helix, an active site lid that controls the product release. The C-terminal is extended by a dynamical single α-helix (SAH), which improves the product release rate by up to ~8-fold, and the overall catalytic rate kcat by up to ~2-fold. It is also shown that the kcat increase is due to the favorable activation entropy change. The NMR H/D exchange data indicate that the conformational dynamics of the transition state analog complex increases as the helix is extended, elucidating the origin of the enhanced catalytic entropy. This study highlights a novel dynamics engineering strategy that can accelerate the overall catalysis through the entropy-driven mechanism.

Self-organization of zinc ions with a photosensitizer in vivo for enhanced antibiofilm and infected wound healing.

Antimicrobial materials have been developed to combat bacteria more effectively and promote infected wound healing. However, it is widely recognized that the potential toxic effects and complexity of the synthesis process hinder their practical applications. In this work, we introduced a strategy for fighting bacteria and promoting wound healing caused by Staphylococcus epidermidis (S. epidermidis) infection by the self-combination of Zn2+ and clinically applied 5-aminolevulinic acid hydrochloride (ALA) in the microbes. The clinical ALA could target and accumulate in the biofilm as well as contribute to the low-dose Zn2+ penetrating the biofilm due to the self-organized formation of Zn protoporphyrin IX in situ. Upon exposing to a 635 nm laser, the self-combination of ALA and Zn2+ significantly inhibited and eliminated the S. epidermidis biofilm via a synergistic biofilm eradication mechanism that enhanced photodynamic inactivation and aggravated cell wall/membrane disruption. In addition, the combination of ALA and Zn2+ could accelerate wound repair and reduce inflammatory response without causing cytotoxicity. The proposed strategy in this study illustrates the clinical prospects of eradicating biofilms and repairing infected wounds and demonstrates good biocompatibility towards infectious diseases.

Corrigendum to "Vanillic Acid Alleviates Acute Myocardial Hypoxia/Reoxygenation Injury by Inhibiting Oxidative Stress".

[This corrects the article DOI: 10.1155/2020/8348035.].

Loss of Nobox prevents ovarian differentiation from juvenile ovaries in zebrafish.

As a species without master sex-determining genes, zebrafish displays high plasticity in sex differentiation, making it an excellent model for studying the regulatory mechanisms underlying gonadal differentiation and gametogenesis. Despite being a gonochorist, zebrafish is a juvenile hermaphrodite that undergoes a special phase of juvenile ovary before further differentiation into functional testis and ovary. The mechanisms underlying juvenile ovary formation and subsequent gonadal differentiation remain largely unknown. In this study, we explored the role of Nobox/nobox (new born ovary homeobox protein), another oocyte-specific transcription factor in females, in early zebrafish gonadogenesis using CRISPR/Cas9 technology. As in mammals, nobox is specifically expressed in zebrafish gonads with a dimorphic pattern at juvenile stage. In contrast to the mutant of figla (factor in the germline alpha, another oocyte-specific transcription factor), the nobox mutants showed formation of typical perinucleolar (PN) follicles at primary growth (PG) stage in juvenile gonads, suggesting occurrence of follicle assembly from cystic oocytes (chromatin nucleolar stage, CN). These follicles, however, failed to develop further to form functional ovaries, resulting in all-male phenotype. Despite its expression in adult testis, the loss of nobox did not seem to affect testis development, spermatogenesis and male spawning. In summary, our results indicate an important role for Nobox in zebrafish ovarian differentiation and early folliculogenesis.

Liquiritigenin enhances cyclic adenosine monophosphate production to mitigate inflammation in dendritic cells.

OBJECTIVE: This study aims to dissect the mechanism of traditional Chinese medicinal herbs against asthma; we chose to first focus on the main chemical components of licorice to investigate their contribution to asthmatic inflammation inhibition. METHODS: Production of cellular nucleotide molecules such as cAMP, cGMP, and cGAMP was examined by using enzyme-linked immunosorbent assay (ELISA). Enzyme-encoding genes were tested in vitro using quantitative real-time PCR and protein level was detected by Western blotting analysis. In addition, co-culturing of murine dendritic cells together with T cells was conducted to examine the expression of cytokine genes and host immune response. RESULTS: We found that one of the components within licorice, named liquiritigenin (LR), could efficiently enhance cAMP production in different cell lines. The augmentation of such molecules was linked to the high expression of cAMP synthesis genes and repressed expression of cAMP breaking down genes. In addition, the downstream immune response was also alleviated by the increase in cAMP levels by LR, suggesting the great potential of this molecule against inflammation. Subsequent immunological tests showed that LR could efficiently inhibit the expression of several cytokines and alter the NF-κB pathway and T cell polarization. CONCLUSION: Altogether, we have identified a promising antiasthmatic agent LR that could exhibit immunosuppressive function by elevating the cAMP level.

Xanthoceraside induces cell apoptosis through downregulation of the PI3K/Akt/Bcl-2/Bax signaling pathway in cell lines of human bladder cancer.

BACKGROUND: Xanthoceraside is a component obtained in the husks of Xanthoceras sorbifolia Bunge. Series of researches proved that xanthoceraside had functions of anti-inflammation and anti-tumor effects. However, the mechanisms of xanthoceraside against bladder cancer are unclear. Accordingly, we proposed to investigate xanthoceraside's impacts and potential mechanisms in cells of bladder cancer. METHODS: By using the CCK-8 assay, we measured the viability of cells. With the use of 4,6-diamidino-2-phenylindole (DAPI) staining, we examined nuclear fragmentation and chromatin condensation in the nuclei of apoptotic cells. By using flow cytometry, we measured cell apoptosis. By using Western blotting, we tested the expressions of Caspase-9, Caspase-8, Caspase-3, Bcl-xL, P53, and PI3K/Akt/Bcl-2/Bax. RESULTS: The proliferation of cell lines of human bladder cancer T24 and 5637 was suppressed by xanthoceraside significantly in a time- and concentration-dependent way. When cell lines 5637 and T24 were incubated as the xanthoceraside dose increased, the rates of cell apoptosis were upregulated, which was dependent on dose. According to further analysis, xanthoceraside induced apoptosis by upregulating Bax and downregulating the expression of Bcl-xL and Bcl-2. However, xanthoceraside did not change the expression of Caspase-9, Caspase-8, and Caspase-3. Interestingly, xanthoceraside also downregulated the expression of p-PI3K and p-Akt, and upregulated P53. CONCLUSIONS: Xanthoceraside induces cell apoptosis through downregulation of the PI3K/Akt/Bcl-2/Bax signaling pathway in cell lines of human bladder cancer.

Circular RNA circ_0003645 silencing alleviates inflammation and apoptosis via the NF-κB pathway in endothelial cells induced by oxLDL.

Atherosclerosis (AS) is a serious threat to the cardiovascular system. Circular RNA circ_0003645 was found to be differentially expressed in the process of AS. Our study tried to unravel the effect and underlying mechanism of circ_0003645 in endothelial cells treated with oxidized low-density lipoprotein (oxLDL). Si-RNAs and over-circ0003645 were transfected into human umbilical vein endothelial cells (HUVECs), and the expression levels of circ_0003645 and NF-κB mRNA were measured. The protein level of NF-κB, lactate dehydrogenase leakage (LDH leakage), cell viability, and apoptosis were detected. Further, the expression of interleukin (IL)-6, tumor necrosis factor (TNF)-α, ICAM-1, and VCAM-1 were measured. Circ_0003645 was found up-regulated in AS patients and in HUVECs treated with oxLDL. The LDH leakage, cell apoptosis, and expression levels of IL-6, TNF-α, ICAM-1, VCAM-1, NF-κB mRNA, NF-κB protein were all inhibited by circ_0003645 silencing, while cell viability was promoted, and the opposite effects were observed by the overexpression of circ_0003645. In conclusion, circ_0003645 silencing alleviated inflammation and apoptosis, while promoted the viability in oxLDL-induced endothelial cells by the NF-κB pathway.

Vanillic Acid Alleviates Acute Myocardial Hypoxia/Reoxygenation Injury by Inhibiting Oxidative Stress.

Oxidative stress is an important factor of myocardial hypoxia/reoxygenation (H/R) injury. Our research focuses on how to reduce the cardiac toxicity caused by oxidative stress through natural plant extracts. Vanillic acid (VA) is a phenolic compound found in edible plants and rich in the roots of Angelica sinensis. Experimental studies have provided evidence for this compound's effectiveness in cardiovascular diseases; however, its mechanism is still unclear. In this study, molecular mechanisms related to the protective effects of VA were investigated in H9c2 cells in the context of H/R injury. The results showed that pretreatment with VA significantly increased cell viability and decreased the percentage of apoptotic cells, as well as lactate dehydrogenase and creatine phosphokinase activity, in the supernatant, accompanied by reduced levels of reactive oxygen species and reduced caspase-3 activity. VA pretreatment also restored mitochondrial membrane potentials. Moreover, preincubation with VA significantly attenuated mitochondrial permeability transition pore activity. VA administration upregulated adenosine monophosphate-activated protein kinase α2 (AMPKα2) protein expression, and interestingly, pretreatment with AMPKα2-siRNA lentivirus effectively attenuated the cardioprotective effects of VA in response to H/R injury.

Substrate Recognition and Catalytic Mechanism of the Phosphate Acyltransferase PlsX from Bacillus subtilis.

Phosphate: acyl-acyl carrier protein (ACP) acyltransferase PlsX is a peripheral enzyme catalysing acyl transfer to orthophosphate in phospholipid synthesis. Little is known about how it recognises substrates and catalyses the acyl transfer. Here we show that its active site includes many residues lining a long, narrow gorge at the dimeric interface, two positive residues forming a positive ACP docking pad next to the interfacial gorge, and a number of strictly conserved residues significantly contributing to the catalytic activity. These findings suggest a substrate recognition mode and a catalytic mechanism that are different from those of phosphotransacetylases catalysing a similar acyl transfer reaction. The catalytic mechanism involves substrate activation and transition-state stabilization by two strictly conserved residues, Lys184 and Asn229. Another noticeable feature of the catalysis is the release of the acyl phosphate product near the membrane, which might facilitate its membrane insertion.

[Let-7i reduces chemotherapy resistance in breast cancer cells through down-regulation of K-Ras and Bcl2 expression].

Objective To investigate the effect of microRNA let-7i on the drug-resistant cells of human breast cancer and its potential molecular mechanisms. Methods Reverse transcription PCR was performed to detect the relative levels of let-7i expressed in breast cancer cell line MCF-7 as well as MCF-7 resistant to both cisplatin (CDDP) and adriamycin (ADR) (MCF-7/CDDP, MCF-7/ADR) cells. CCK-8 assay was used to measure the varied sensitivity of cell MCF-7, MCF-7/CDDP and MCF-7/ADR to ADR following chemotherapy. CCK-8 assay and colony formation assay were carried out to observe the change of sensitivity to ADR after let-7i was over-expressed or inhibited, and flow cytometry was used to determine the induced apoptosis of breast cancer cells resistant to ADR following over-expression of let-7i. ELISA was conducted to measure the activity of caspase-3/9 against breast cancer drug. Reverse transcription-PCR was performed to detect the mRNA levels of Bcl2 and K-Ras, and Western blot analysis to examine the protein levels of Bcl2, BAX and K-Ras. Results Let-7i was down-regulated in drug-resistant breast cancer cells as compared with simple MCF-7 cell line, and negatively correlated with chemotherapy resistance. Compared with negative control group, over-expression of let-7i resulted in improved cell viability and colony formation of drug-resistant breast cancer cells, and facilitated the cancer cell apoptosis induced by ADR, yet raised caspase-3/9 activity. Bcl2 level decreased, whereas BAX increased with added ADR concentration. Over-expressed let-7i significantly inhibited Bcl2 and K-Ras expression in the drug-resistant cells. Conclusion Let-7i is down-regulated in drug-resistant breast cancer cells, and negatively correlated with chemotherapy resistance. The findings suggest that let-7i may inhibit the resistance of drug-resistant breast cancer cells via inhibiting the expression of K-Ras and Bcl2.

Identification of an amphipathic peptide sensor of the Bacillus subtilis fluid membrane microdomains.

Regions of increased fluidity are newly found bacterial membrane microdomains that are composed of short, unsaturated and branched fatty acyl chains in a fluid and disordered state. Currently, little is known about how proteins are recruited and localized to these membrane domains. Here, we identify a short amphipathic α-peptide in a previously unreported crystal structure and show that it is responsible for peripheral localization of the phosphate acyltransferase PlsX to the fluid microdomains in Bacillus subtilis. Mutations disrupting the amphipathic interaction or increasing the nonpolar interaction are found to redistribute the protein to the cytosol or other part of the plasma membrane, causing growth defects. These results reveal a mechanism of peripheral membrane sensing through optimizing nonpolar interaction with the special lipids in the microdomains. This finding shows that the fluid membrane microdomains may take advantage of their unique lipid environment as a means of recruiting and organizing proteins.

Baseline Serum Cystatin C Is a Potential Predictor for Acute Kidney Injury in Patients with Acute Pancreatitis.

AIMS: The study is aimed at studying the incidence of acute kidney injury (AKI) and exploring the potential predictor for AKI in patients with acute pancreatitis. METHODS: A retrospective study adopting a stratified cohort sampling design was performed in a cohort of patients (n = 237) diagnosed with acute pancreatitis without any renal injury. The following information including age, gender, serum creatinine, serum urea nitrogen, serum uric acid, serum cystatin C, fasting serum glucose, serum amylase, serum lipase, serum choline esterase, total protein, albumin, globulin, total bilirubin, direct bilirubin, total bile acids, glutamic-pyruvic transaminase, glutamic-oxaloacetic transaminase, gamma glutamyl transpeptidase, and alkaline phosphatase were collected from each patient when they were diagnosed with acute pancreatitis. Student t-test was conducted to figure out the difference between patients with and without AKI. Univariate and multivariate logistic regression analyses were used for investigating the predictors for AKI in patients with acute pancreatitis. RESULTS: 18 (7.6%) patients in total had developed AKI among the study group. Compared with patients without AKI (1.01 ± 0.26 mg/L), the level of baseline serum cystatin C (CYS-C) was significantly higher in patients with AKI (3.64 ± 2.17 mg/L, P < 0.001). Baseline serum CYS-C (OR = 203.594, P < 0.001) was the independent and significant predictor for AKI in patients with acute pancreatitis. AKI in patients with acute pancreatitis could be identified with a sensitivity of 88.9% at specificity of 100% (AUC = 0.948, 95% CI 0.879-1.000) by baseline serum CYS-C (cut-off value = 1.865 mg/L). CONCLUSIONS: Baseline serum CYS-C shall be adopted to predict the potential risk of AKI in patients with acute pancreatitis.

Two active site arginines are critical determinants of substrate binding and catalysis in MenD: a thiamine-dependent enzyme in menaquinone biosynthesis.

The bacterial enzyme MenD, or 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexene-1-carboxylate (SEPHCHC) synthase, catalyzes an essential Stetter reaction in menaquinone (vitamin K2) biosynthesis via thiamine diphosphate (ThDP)-bound tetrahedral post-decarboxylation intermediates. The detailed mechanism of this intermediate chemistry, however, is still poorly understood, but of significant interest given that menaquinone is an essential electron transporter in many pathogenic bacteria. Here, we used site-directed mutagenesis, enzyme kinetic assays, and protein crystallography to reveal an active-inactive intermediate equilibrium in MenD catalysis and its modulation by two conserved active site arginine residues. We observed that these conserved residues play a key role in shifting the equilibrium to the active intermediate by orienting the C2-succinyl group of the intermediates through strong ionic hydrogen bonding. We found that when this interaction is moderately weakened by amino acid substitutions, the resulting proteins are catalytically competent with the C2-succinyl group taking either the active or the inactive orientation in the post-decarboxylation intermediate. When this hydrogen-bonding interaction was strongly weakened, the succinyl group was re-oriented by 180° relative to the native intermediate, resulting in the reversal of the stereochemistry at the reaction center that disabled catalysis. Interestingly, this inactive intermediate was formed with a distinct kinetic behavior, likely as a result of a non-native mode of enzyme-substrate interaction. The mechanistic insights gained from these findings improve our understanding of the new ThDP-dependent catalysis. More importantly, the non-native-binding site of the inactive MenD intermediate uncovered here provides a new target for the development of antibiotics.

Roles of Figla/figla in Juvenile Ovary Development and Follicle Formation During Zebrafish Gonadogenesis.

Sex determination and differentiation are complex processes. As a juvenile hermaphrodite or undifferentiated gonochorist, zebrafish undergo a special juvenile ovarian phase during sex differentiation, making it an excellent model for studying early oogenesis and folliculogenesis. We provide lines of evidence at morphological, molecular, and genetic levels for roles of factor in the germline α (Figla), an oocyte-specific transcription factor, in early zebrafish gonadogenesis. As in mammals, Figla/figla was also expressed in the gonads and its expression in the ovary was also restricted to early oocytes. Disruption of figla gene by CRISPR/Cas9 led to an all-male phenotype in the mutant. Detailed analysis of early gonadal development showed that the germ cells in the mutant were clustered in cysts and underwent meiosis, forming oocytes at prefollicular chromatin nucleolar (CN) stage (stage IA). However, the subsequent transition from cystic CN oocytes to individual follicular perinucleolar oocytes (stage IB) was blocked, resulting in an all-male phenotype in the mutant. The phenotype of figla mutant could not be rescued by estrogen treatment, in contrast to cyp19a1a mutant, and introduction of tp53 mutation also had no effect, unlike in fancd1 and fancl mutants. Transcriptome analysis revealed that many biological processes and pathways related to germ cell development, especially oogenesis, were upregulated in the presence of Figla and that the regulation of figla expression may involve heat shock proteins. Our results strongly suggest important roles for Figla in juvenile ovary development, especially the formation of individual follicles from cystic oocytes.