Mitigation of Salt Stress in Rice by the Halotolerant Plant Growth-Promoting Bacterium Enterobacter asburiae D2.

Salinity is a major abiotic stress that seriously affects crop growth worldwide. In this work, we aimed to isolate potential halotolerant plant growth-promoting rhizobacteria (PGPR) to mitigate the adverse impacts of salt stress in rice. An isolate, D2, with multiple plant growth-promoting (PGP) characteristics was identified as Enterobacter asburiae D2. Strain D2 could produce indole-3-acetic acid and siderophore. It also exhibited phosphate solubilization and 1-aminocyclopropane-1-carboxylic deaminase activity. Genome analysis further provided insights into the molecular mechanism of its PGP abilities. Strain D2 inoculation efficiently stimulated rice growth under both normal and saline conditions. Compared with the non-inoculated plants, a significant increase in plant height (18.1-34.7%), root length (25.9-57.1%), root dry weight (57.1-150%), and shoot dry weight (17.3-50.4%) was recorded in inoculated rice seedlings. Meanwhile, rice seedlings inoculated with strain D2 showed improvement in chlorophyll and proline content, while the oxidant damage was reduced in these plants in comparison with the control group. Moreover, the K+/Na+ ratio of the inoculated rice seedlings exposed to NaCl and Na2CO3 was higher than that of the uninoculated groups. These results imply that Enterobacter asburiae D2 is a potential PGPR that can be used for alleviation of salt stress in rice.

hCINAP regulates the differentiation of embryonic stem cells by regulating NEDD4 liquid-liquid phase-separation-mediated YAP1 activation.

YAP1 functions in lineage differentiation of pluripotent embryonic stem cells (ESCs); however, the detailed mechanisms underlying the regulation of YAP1 activity during ESC differentiation remain elusive. Here, we report that hCINAP serves as a negative regulator of YAP1 during ESC fate decisions. The expression of mCINAP, the murine homolog of hCINAP, is downregulated during the differentiation process of murine ESC (mESC) ectoderm lineage, leading to liquid-liquid phase separation (LLPS) of NEDD4 and activation of YAP1. Mechanistically, hCINAP interacts with and prevents NEDD4 from forming cytoplasmic condensates that compartmentalize YAP1 and its kinase NLK, facilitating YAP1 phosphorylation at Ser128 and promoting YAP1 activation. mCINAP depletion leads to the formation of NEDD4 condensates and YAP1 activation, which impedes endoderm differentiation of mESCs. Our study shows that hCINAP is a vital regulator of YAP1 activity and is essential for stem cell fate decisions, which provides mechanistic insight into early embryogenesis.

Ultrafast synthesis of laccase-copper phosphate hybrid nanoflowers for efficient degradation of tetracycline antibiotics.

The presence of antibiotics in the environment causes increasing attention due to their potential risks to ecosystems and public health. Laccases are versatile oxidases capable of degrading various organic contaminants including pharmaceuticals. However, the performance of bacterial laccases on tetracycline antibiotics (TCs) degradation is seldom investigated. In this work, a bacterial laccase from Bacillus amyloliquefaciens was immobilized as laccase-inorganic hybrid nanoflowers (Lac-hNFs) by a facile and rapid method. The immobilized laccase was employed to remove different TCs including tigecycline, which is a third-generation TC that its degradation by laccase has not been reported. Lac-hNFs were synthesized by sonication-mediated self-assembly of laccase and copper ions in 5 min at room temperature. About 95% of laccase could be encapsulated in the nanoflowers, and the obtained Lac-hNFs exhibited great enhancement in stability under harsh conditions. The immobilized laccase showed a half-life of 11.7 h at 60 °C, which was about 1.4-fold higher than that of the free enzyme. Meanwhile, Lac-hNFs retained 81% of the initial activity after incubation at 25 °C for 10 days. The laccase in combination with acetosyringone could efficiently decompose tetracycline, doxycycline, and tigecycline. More than 79% of the three TCs were transformed in 1 h. Compared with the free enzyme, Lac-hNFs demonstrated higher capacity in the removal of TCs. Furthermore, Lac-hNFs remained their high degradation capacity after five cycles of reuse. Bacterial growth inhibition test revealed that most of the toxicity of TCs was eliminated after Lac-hNFs treatment. The main transformation products were identified by LC-MS, and the possible degradation pathways were proposed. The interaction mechanism between laccase and TCs was also analyzed using molecular docking. This work provides an efficient way to remove toxic organic pollutants.

Deubiquitinase OTUD6A promotes breast cancer progression by increasing TopBP1 stability and rendering tumor cells resistant to DNA-damaging therapy.

The DNA damage response (DDR) is critical for maintaining cellular homeostasis and genome integrity. Mounting evidence has shown that posttranslational protein modifications play vital roles in the DDR. In this study, we showed that deubiquitinase OTUD6A is involved in the DDR and is important for maintaining genomic stability. Mechanistically, in response to DNA damage, the abundance of OTUD6A was increased; meanwhile, PP2A interacted with OTUD6A and dephosphorylated OTUD6A at sites S70/71/74, which promoted nuclear localization of OTUD6A. Subsequently, OTUD6A was recruited to the damage site, where it interacted with TopBP1 and blocked the interaction between TopBP1 and its ubiquitin E3 ligase UBR5, decreasing K48-linked polyubiquitination and increasing the stability of TopBP1. OTUD6A depletion impaired CHK1 S345 phosphorylation and blocked cell cycle progression under DNA replication stress. Consistently, knockout of OTUD6A rendered mice hypersensitive to irradiation, shortened survival, and inhibited tumor growth by regulating TopBP1 in xenografted nude mice. Moreover, OTUD6A is expressed at high levels in breast cancer, and OTUD6A overexpression promotes cell proliferation, migration and invasion, indicating that dysregulation of OTUD6A expression contributes to genomic instability and is associated with tumor development. In summary, this study demonstrates that OTUD6A plays a critical role in promoting tumor cell resistance to chemoradiotherapy by deubiquitinating and stabilizing TopBP1.

Deubiquitinase OTUB2 exacerbates the progression of colorectal cancer by promoting PKM2 activity and glycolysis.

Aberrant regulation of ubiquitination often leads to metabolic reprogramming in tumor cells. However, the underlying mechanisms are not fully understood. Here we demonstrate that OTUB2, an OTU deubiquitinase, is upregulated in colorectal cancer (CRC) and exacerbates the progression of CRC through modulating the aerobic glycolysis. Mechanistically, OTUB2 directly interacts with pyruvate kinase M2 (PKM2) and inhibits its ubiquitination by blocking the interaction between PKM2 and its ubiquitin E3 ligase Parkin, thereby enhancing PKM2 activity and promoting glycolysis. In response to glucose starvation stress, the effect of OTUB2 on PKM2 is enhanced, which confers metabolic advantage to CRC cells. Moreover, OTUB2 depletion reduces glucose consumption, lactate production, and cellular ATP production. OTUB2-knockout CRC cells exhibit attenuated proliferation and migration, as well as an elevated level of apoptosis and increased sensitivity to chemotherapy drugs. Furthermore, in vivo assays show that knockout of OTUB2 inhibits tumor growth in mice. Taken together, these findings reveal the critical role of OTUB2 in the regulation of glycolysis and illustrate the molecular mechanism underlying its role as a negative regulator of PKM2 ubiquitination in CRC, establishing a bridge between OTUB2-regulated PKM2 ubiquitination and altered metabolic patterns in CRC and suggesting that OTUB2 is a promising target for CRC treatment.

Preparation and synthetic dye decolorization ability of magnetic cross-linked enzyme aggregates of laccase from Bacillus amyloliquefaciens.

Laccases are versatile oxidases that are capable of decolorizing various synthetic dyes. Recombinant Bacillus amyloliquefaciens laccase was immobilized as magnetic cross-linked enzyme aggregates (M-CLEAs) for application in dye decolorization. Several parameters influencing the activity recovery were evaluated during the synthesis of M-CLEAs. With ammonium sulfate as precipitant, maximum activity was recovered by cross-linking with 0.16% glutaraldehyde for 1 h. The prepared M-CLEAs exhibited improved activity under alkaline conditions. It remained 74% activity after incubation at 60 °C for 5 h. Enhanced tolerance towards NaCl was also observed for the M-CLEAs, with 68% activity remaining in the presence of 1 M NaCl. The immobilized laccase could rapidly decolorize more than 93% of reactive black 5 and indigo carmine in 1 h, while its catalytic efficiency towards reactive blue 19 was relatively low. After four cycles of consecutive reuse, the M-CLEAs could decolorize 92% of indigo carmine. The easy recovery and reusability of M-CLEAs facilitate the potential application of bacterial laccase in dye decolorization.

High-level expression of Bacillus amyloliquefaciens laccase and construction of its chimeric variant with improved stability by domain substitution.

Large-scale application of bacterial laccases is usually limited by their low production, and their recombinant expression in Escherichia coli is prone to form inactive aggregates in the cytoplasm. In this work, we optimized the expression conditions of Bacillus amyloliquefaciens laccase (LacA) in E. coli, and obtained high yield for the extracellular production of LacA. The final activity reached 20,255 U/L for LacA, which is among one of the highest activities for recombinant bacterial laccases. Moreover, a chimeric enzyme (Lac3A/S) was designed based on LacA by domain substitution with a stable laccase from B. subtilis. The hybrid laccase could also be secreted into the culture medium with high expression level, and had higher thermal and alkaline stabilities than those of LacA. It was fully active after 10-day incubation at pH 9.0, and retained 47% of its initial activity after incubation at 70 °C for 5 h. Homology analysis of protein structure indicated Lac3A/S had a more packed structure in the copper-binding sites than LacA, which might lead to an enhancement in stability under harsh conditions.

The Deubiquitinase USP38 Promotes NHEJ Repair through Regulation of HDAC1 Activity and Regulates Cancer Cell Response to Genotoxic Insults.

The DNA damage response (DDR) is essential for maintaining genome integrity. Mounting evidence reveals that protein modifications play vital roles in the DDR. Here, we show that USP38 is involved in the DDR by regulating the activity of HDAC1. In response to DNA damage, USP38 interacted with HDAC1 and specifically removed the K63-linked ubiquitin chain promoting the deacetylase activity of HDAC1. As a result, HDAC1 was able to deacetylate H3K56. USP38 deletion resulted in persistent focal accumulation of nonhomologous end joining (NHEJ) factors at DNA damage sites and impaired NHEJ efficiency, causing genome instability and sensitizing cancer cells to genotoxic insults. Knockout of USP38 rendered mice hypersensitive to irradiation and shortened survival. In addition, USP38 was expressed at low levels in certain types of cancers including renal cell carcinoma, indicating dysregulation of USP38 expression contributes to genomic instability and may lead to tumorigenesis. In summary, this study identifies a critical role of USP38 in modulating genome integrity and cancer cell resistance to genotoxic insults by deubiquitinating HDAC1 and regulating its deacetylation activity. SIGNIFICANCE: This study demonstrates that USP38 regulates genome stability and mediates cancer cell resistance to DNA-damaging therapy, providing insight into tumorigenesis and implicating USP38 as a potential target for cancer diagnosis.

hCINAP regulates the DNA-damage response and mediates the resistance of acute myelocytic leukemia cells to therapy.

Acute myeloid leukemia (AML) is a genetically heterogeneous malignant disorder of the hematopoietic system, characterized by the accumulation of DNA-damaged immature myeloid precursors. Here, we find that hCINAP is involved in the repair of double-stranded DNA breaks (DSB) and that its expression correlates with AML prognosis. Following DSB, hCINAP is recruited to damage sites where it promotes SENP3-dependent deSUMOylation of NPM1. This in turn results in the dissociation of RAP80 from the damage site and CTIP-dependent DNA resection and homologous recombination. NPM1 SUMOylation is required for recruitment of DNA repair proteins at the early stage of DNA-damage response (DDR), and SUMOylated NPM1 impacts the assembly of the BRCA1 complex. Knockdown of hCINAP also sensitizes a patient-derived xenograft (PDX) mouse model to chemotherapy. In clinical AML samples, low hCINAP expression is associated with a higher overall survival rate in patients. These results provide mechanistic insight into the function of hCINAP during the DNA-damage response and its role in AML resistance to therapy.

NEDDylation antagonizes ubiquitination of proliferating cell nuclear antigen and regulates the recruitment of polymerase η in response to oxidative DNA damage.

NEDDylation has been shown to participate in the DNA damage pathway, but the substrates of neural precursor cell expressed developmentally downregulated 8 (NEDD8) and the roles of NEDDylation involved in the DNA damage response (DDR) are largely unknown. Translesion synthesis (TLS) is a damage-tolerance mechanism, in which RAD18/RAD6-mediated monoubiquitinated proliferating cell nuclear antigen (PCNA) promotes recruitment of polymerase η (polη) to bypass lesions. Here we identify PCNA as a substrate of NEDD8, and show that E3 ligase RAD18-catalyzed PCNA NEDDylation antagonizes its ubiquitination. In addition, NEDP1 acts as the deNEDDylase of PCNA, and NEDP1 deletion enhances PCNA NEDDylation but reduces its ubiquitination. In response to H2O2 stimulation, NEDP1 disassociates from PCNA and RAD18-dependent PCNA NEDDylation increases markedly after its ubiquitination. Impairment of NEDDylation by Ubc12 knockout enhances PCNA ubiquitination and promotes PCNA-polη interaction, while up-regulation of NEDDylation by NEDD8 overexpression or NEDP1 deletion reduces the excessive accumulation of ubiquitinated PCNA, thus inhibits PCNA-polη interaction and blocks polη foci formation. Moreover, Ubc12 knockout decreases cell sensitivity to H2O2-induced oxidative stress, but NEDP1 deletion aggravates this sensitivity. Collectively, our study elucidates the important role of NEDDylation in the DDR as a modulator of PCNA monoubiquitination and polη recruitment.

Brugada phenocopy in a patient with acute pulmonary embolism presenting with recurrent syncope.

Brugada phenocopy (BrP) refers to a group of clinical conditions that have etiologies distinct from Brugada syndrome (BrS). Although both demonstrate features of ST-segment elevation in the right precordial leads on the electrocardiogram (ECG), one must be distinguished from the other as their treatment options are different. We report a male patient who presented with recurrent syncope with a Brugada and a S1Q3T3 pattern on the ECG. Acute pulmonary embolism (APE) complicated by BrS was suspected. Twenty-four hours Holter monitoring did not demonstrate any evidence of ventricular arrhythmias. Computed tomography pulmonary angiogram confirmed the presence of an APE. He was treated with low molecular weight heparin and a repeat ECG taken the next day showed resolution of the Brugada and S1Q3T3 patterns. This case report illustrates that APE and BrS can present with similar clinical and electrocardiographic features of recurrent syncope and Brugada pattern, respectively.

Preparation and characterization of Zr-N-codoped TiO2 nano-photocatalyst and its activity enhanced-mechanism.

Zr-N-codoped TiO2 nano-photocatalyst was prepared through sol-gel method using ammonia water and zirconium nitrate as the source of N and Zr, respectively. The resulting materials were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS) and ultraviolet-visible diffuse reflectance spectroscopy (UV-vis DRS). XRD results showed that codoping with Zr and N elements could greatly inhibit the phase transformation of TiO2 from anatase to rutile. XPS analysis indicated that Zr4+ was incorporated into the lattice of TiO2 through substituting titanium atoms. Meanwhile, N was also incorporated into the lattice of TiO2 through substituting oxygen atoms and existed in the form of N-Ti-O. DRS revealed that the light absorption edge of Zr-N-TiO2 was significantly red-shifted to visible region, leading to a narrower band gap and higher visible photocatalytic activity. The enhanced visible activity was attributed to the well anatase crystallite, intense light absorbance in visible region and narrow band gap.

Effect of endogenous hydrolytic enzymes pretreatment on the anaerobic digestion of sludge.

In this study, the effects of endogenous amylase, endogenous protease and combined amylase/protease pretreatment of sludge were studied to enhance the efficiency of sludge anaerobic digestion. These enzymes were obtained from bacterial fermentation and bacteria were separated from the sludge. All treatments improved sludge solubilization and acidification but had little influence on the floc sizes. In terms of sludge solubilization and acidification amylase was better than protease or mixed enzyme. After 7 h endogenous amylase treatment, the supernatant soluble chemical oxygen demand and volatile fatty acids concentration increased by 78.2% and 129.6%, respectively. But, in terms of anaerobic biodegradability, the best result was obtained with combined enzyme treatment, biogas production increased by 23.1% compared to the control after 11 days of anaerobic digestion. Scanning electron micrographs observation and particle size analysis revealed that the most important mechanism for the enzyme treatment of sludge might be solubilization of extracellular polymeric substances.

Purification and characterization of laccase from Pycnoporus sanguineus and decolorization of an anthraquinone dye by the enzyme.

The white rot fungus Pycnoporus sanguineus produced high amount of laccase in the basal liquid medium without induction. Laccase was purified using ultrafiltration, anion-exchange chromatography, and gel filtration. The molecular weight of the purified laccase was estimated as 61.4 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme oxidized typical substrates of laccases including 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate), 2,6-dimethoxyphenol, and syringaldazine. The optimum pH and temperature for the purified laccase were 3.0 and 65 degrees C, respectively. The enzyme was stable up to 40 degrees C, and high laccase activity was maintained at pH 2.0-5.0. Sodium azide, L-cysteine, and dithiothreitol strongly inhibited the laccase activity. The purified enzyme efficiently decolorized Remazol Brilliant Blue R in the absence of added redox mediators. The high production of P. sanguineus laccase as well as its decolorization ability demonstrated its potential applications in dye decolorization.