Improvement in Salt Tolerance Ability of Pseudomonas putida KT2440.

Pseudomonas putida KT2440 is a popular platform for bioremediation due to its robust tolerance to environmental stress and strong biodegradation capacity. Limited research on the salt tolerance of P. putida KT2440 has hindered its application. In this study, the strain KT2440 was tested to tolerate a maximum of 4% w/v NaCl cultured with minimal salts medium. Transcriptomic data in a high-salinity environment showed significant expression changes in genes in membrane components, redox processes, chemotaxis, and cellular catabolic processes. betB-encoding betaine-aldehyde dehydrogenase was identified from the transcriptome data to overexpress and enhance growth profile of the strain KT2440 in minimal salts medium containing 4% w/v NaCl. Meanwhile, screening for exogenous salt-tolerant genes revealed that the Na+/H+ antiporter EcnhaA from Escherichia coli significantly increased the growth of the strain KT2440 in 4% w/v NaCl. Then, co-expression of EcnhaA and betB (KT2440-EcnhaA-betB) increased the maximum salt tolerance of strain KT2440 to 5% w/v NaCl. Further addition of betaine and proline improved the salt tolerance of the engineered strain to 6% w/v NaCl. Finally, the engineered strain KT2440-EcnhaA-betB was able to degrade 56.70% of benzoic acid and 95.64% of protocatechuic acid in minimal salt medium containing 4% w/v NaCl in 48 h, while no biodegradation was observed in the normal strain KT2440 in the same conditions. However, the strain KT2440-EcnhaA-betB failed to degrade catechol in minimal salt medium containing 3% w/v NaCl. This study illustrated the improvement in the salt tolerance performance of Pseudomonas putida KT2440 and the feasibility of engineered strain KT2440 as a potential salt-tolerant bioremediation platform.

MST1/2 regulates fibro/adipogenic progenitor fate decisions in skeletal muscle regeneration.

Defective skeletal muscle regeneration is often accompanied by fibrosis. Fibroblast/adipose progenitors (FAPs) are important in these processes, however, the regulation of FAP fate decisions is unclear. Here, using inducible conditional knockout mice, we show that blocking mammalian Ste20-like kinases 1/2 (MST1/2) of FAPs prevented apoptosis and reduced interleukin-6 secretion in vivo and in vitro, which impaired myoblast proliferation and differentiation, as well as impaired muscle regeneration. Deletion of Mst1/2 increased co-localization of Yes-associated protein (YAP) with Smad2/3 in nuclei and promoted differentiation of FAPs toward myofibroblasts, resulting in excessive collagen deposition and skeletal muscle fibrosis. Meanwhile, inhibition of MST1/2 increased YAP/Transcriptional co-activator with PDZ-binding motif activation, which promoted activation of the WNT/ß-catenin pathway and impaired the differentiation of FAPs toward adipocytes. These results reveal a new mechanism for MST1/2 action in disrupted skeletal muscle regeneration and fibrosis via regulation of FAP apoptosis and differentiation. MST1/2 is a potential therapeutic target for the treatment of some myopathies.

Inactivation of MST1/2 Controls Macrophage Polarization to Affect Macrophage-Related Disease via YAP and Non-YAP Mechanisms.

Macrophage polarization is a critical process that regulates in inflammation, pathogen defense, and tissue repair. Here we demonstrate that MST1/2, a core kinase of Hippo pathway and a recently identified regulator of inflammation, plays a significant role in promoting M2 polarization. We provide evidence that inhibition of MST1/2, achieved through either gene-knockout or pharmacological treatment, leads to increased M1 polarization in a YAP-dependent manner, resulting in the development of M1-associated inflammatory disorders. Moreover, MST1/2 inhibition also leads to a substantial reduction in M2 polarization, but this occurs through the STAT6 and MEK/ERK signaling. The STAT6 is independent of YAP, but MEK/ERK is dependent of YAP. Consistent with these observations, both MST1/2-conditional knockout mice and wild-type mice treated with XMU-MP-1, a chemical inhibitor of MST1/2, exhibited reduced M2-related renal fibrosis, while simultaneously displaying enhanced LPS-mediated inflammation and improved clearance of MCR3-modified gram-negative bacteria. These findings uncover a novel role of MST1/2 in regulating macrophage polarization and establish it as a potential therapeutic target for the treatment of macrophage-related fibrotic diseases.

Multiscale imaging of peroxynitrite in gliomas with a blood-brain barrier permeable probe reveals its potential as a biomarker and target for glioma treatment.

Cancer development is driven by diverse processes, and metabolic alterations are among the primary characteristics. Multiscale imaging of aberrant metabolites in cancer is critical to understand the pathology and identify new targets for treatment. While peroxynitrite (ONOO-) is reported being enriched in some tumors and plays important tumorigenic roles, whether it is upregulated in gliomas remains unexplored. To determine the levels and roles of ONOO- in gliomas, efficient tools especially those with desirable blood-brain barrier (BBB) permeability and can realize the in situ imaging of ONOO- in multiscale glioma-related samples are indispensable. Herein, we proposed a strategy of physicochemical property-guided probe design, which resulted in the development of a fluorogenic probe NOSTracker for smartly tracking ONOO-. The probe showed sufficient BBB permeability. ONOO- triggered oxidation of its arylboronate group was automatically followed by a self-immolative cleavage of a fluorescence-masking group, liberating its fluorescence signal. The probe was not only highly sensitive and selective towards ONOO-, but its fluorescence favored desirable stability in various complex biological milieus. Guaranteed by these properties, multiscale imaging of ONOO- was realized in vitro in patient-derived primary glioma cells, ex vivo in clinical glioma slices, and in vivo in the glioma of live mice. The results showed the upregulation of ONOO- in gliomas. Furthermore, a specific ONOO- scavenger uric acid (UA) was pharmaceutically used to downregulate ONOO- in glioma cell lines, and an anti-proliferative effect was observed. These results taken together imply the potential of ONOO- as a biomarker and target for glioma treatment, and propose NOSTracker as a reliable tool to further explore the role of ONOO- in glioma development.

The potential role of Hippo pathway regulates cellular metabolism via signaling crosstalk in disease-induced macrophage polarization.

Macrophages polarized into distinct phenotypes play vital roles in inflammatory diseases by clearing pathogens, promoting tissue repair, and maintaining homeostasis. Metabolism serves as a fundamental driver in regulating macrophage polarization, and understanding the interplay between macrophage metabolism and polarization is crucial for unraveling the mechanisms underlying inflammatory diseases. The intricate network of cellular signaling pathway plays a pivotal role in modulating macrophage metabolism, and growing evidence indicates that the Hippo pathway emerges as a central player in network of cellular metabolism signaling. This review aims to explore the impact of macrophage metabolism on polarization and summarize the cell signaling pathways that regulate macrophage metabolism in diseases. Specifically, we highlight the pivotal role of the Hippo pathway as a key regulator of cellular metabolism and reveal its potential relationship with metabolism in macrophage polarization.

Small-Molecule Fluorescent Probes for Detecting HDAC Activity.

Histone deacetylases (HDACs) play critical roles in epigenetic modification. These enzymes can remove acetyl groups from the N-terminal lysine residues of histones, thereby regulating gene expression. Because of their great relevance to various diseases, numerous HDAC inhibitors have been developed. In this context, assays for HDAC activity are prerequisite. Due to the advantages of small-molecule fluorescent probes, researchers have developed many probes to detect HDAC activity for developing HDAC inhibitors. Based on the mechanism of action, two main types of small-molecule fluorescent probes are known. One type is based on binding affinity that are generally HDAC inhibitor-fluorophore conjugates. The other one is enzyme-activated probes, which act as HDAC substrates and show fluorogenic or ratiometric response after being deacetylated by HDACs.

Dynamic control of 4-hydroxyisoleucine biosynthesis by multi-biosensor in Corynebacterium glutamicum.

4-hydroxyisoleucine (4-HIL) has a potential value in treating diabetes. The α-ketoglutarate (α-KG)-dependent isoleucine dioxygenase (IDO) can catalyze the hydroxylation of L-isoleucine (Ile) to form 4-HIL by consuming O2. In our previous study, the ido gene was overexpressed in an Ile-producing Corynebacterium glutamicum strain to synthesize 4-HIL from glucose. Here, a triple-functional dynamic control system was designed to regulate the activity of IDO, the supply of α-KG, O2, and Ile and the synthesis of by-product L-lysine (Lys) for promoting 4-HIL synthesis. Firstly, the codon-optimized ido was positively regulated by seven Ile biosensors Lrp-PbrnFEN with different intensities, and the resulting seven D-NI strains produced 38.7-111.1 mM 4-HIL. Then on the basis of D-NI, odhI and vgb were simultaneously regulated by three PbrnFEN with different intensities to synergistically control α-KG and O2 supply. The 4-HIL titer of twelve D-NINONV strains was more than 90 mM, with D-0I7O7V generating the highest titer of 141.1 ± 15.5 mM. Thirdly, ilvA was negatively regulated by an Ile attenuator PilvBNC on the basis of D-NI strains and some D-NINONV strains to balance the synthesis and conversion of Ile. The resulting D-NIPA strains produced 73.6-123.2 mM 4-HIL, while D-7I7O1VPA accumulated 127.1 ± 20.2 mM 4-HIL. Finally, dapA was negatively regulated by a Lys-OFF riboswitch and Lys content decreased by approximately 70% in most D-RS-NIPA strains. A strain D-RS-5IPA with the highest 4-HIL titer (177.3 ± 8.9 mM) and the lowest Lys concentration (6.1 ± 0.6 mM) was successfully obtained. Therefore, dynamic regulation of main and branch pathway by three functional biosensors can effectively promote 4-HIL biosynthesis in C. glutamicum. KEY POINTS: • Three biosensors were coordinated for dynamic 4-HIL biosynthesis in C. glutamicum • Bidirectional regulation of Ile synthesis and conversion promoted 4-HIL synthesis • Negative regulation of Lys synthesis further increased 4-HIL production.

Enhancing the bioaccessibility of puerarin through the collaboration of high internal phase Pickering emulsions with ß-carotene.

Puerarin, a bioactive flavonoid found in the root of Pueraria lobata, is claimed to possess various medicinal properties. However, application of puerarin in functional foods is currently limited by its poor bioaccessibility. Existing delivery systems that guarantee puerarin bioaccessibility involve complex preparation steps and safety issues. Therefore, this study aimed to use meat protein and olive oil to efficiently and economically fabricate a food grade high internal phase Pickering emulsion (HIPPE) with co-encapsulated puerarin and ß-carotene to improve the bioaccessibility of puerarin. Moreover, the impact on lipid digestibility and puerarin bioaccessibility was verified using a simulated in vitro gastrointestinal tract. Co-encapsulating puerarin and ß-carotene in HIPPE increased puerarin bioaccessibility (85.17%) compared to that achieved with only puerarin in HIPPE (62.66%). This increased bioaccessibility may have been due to the personalized formulation and the exceptional structure of the HIPPE, which slowed down lipid digestion and inhibited puerarin degradation. A synergistic interaction occurred between ß-carotene and HIPPE to improve puerarin bioaccessibility. Our results have important implications for the design of effective delivery systems for encapsulation of puerarin and other bioactive components.

Strategy of De Novo Design toward First-In-Class Imaging Agents for Simultaneously Differentiating Glioma Boundary and Grades.

The extent of resection and tumor grade are two predominant prognostic factors for glioma. Fluorescent imaging is promising to facilitate accurate resection and simultaneous tumor grading. However, no probe fulfilling this task has been reported. Herein, we proposed a strategy of de novo design toward first-in-class fluorescent probes for simultaneously differentiating glioma boundary and grades. By bioinformatics analysis in combination with experimental validation, platelet-derived growth factor receptor ß (PDGFRß) was revealed as a promising biomarker for glioma imaging and grading. Then, fluorogenic probe PDGFP 1 was designed, guided by the structure-activity relationship study. Finally, the probe was demonstrated to stain glioma cells and tissues in the mice orthotopic glioma model with high selectivity over normal brain cells or tissues. Meanwhile, ex vivo experiments using patient-derived samples indicated that the fluorescence was significantly positively correlated with the tumor grades. This result highlighted the feasibility of the three-step de novo probe design strategy and suggested PDGFP 1 as a promising probe for simultaneously differentiating glioma boundary and grades, showing prospects of clinical translation.

Engineering a Bifunctional ComQXPA-PsrfA Quorum-Sensing Circuit for Dynamic Control of Gene Expression in Corynebacterium glutamicum.

Corynebacterium glutamicum is an important industrial workhorse for the production of amino acids and other chemicals. However, the engineering of C. glutamicum is inflexible due to the lack of dynamic regulation tools. In this study, a quorum sensing (QS) circuit and its modulated hfq-sRNA cassette were constructed, and the dynamic control of gene expression by these bifunctional circuits was researched. First, the ComQXPA-PsrfA QS system of Bacillus subtilis was harnessed and modified to create an upregulating QS circuit, in which the transcription of genes controlled by the PsrfA promoter may be promoted at high cell density. This QS circuit successfully activated the expression of green fluorescent protein (GFP) to 6.35-fold in a cell density-dependent manner in C. glutamicum. Next, the hfq-sRNA-mediated downregulating circuit under the control of the ComQXPA-PsrfA QS system was established, and the expression of GFP was autonomously repressed by 96.1%. Next, to fine-tune these two QS circuits, a library of synthetic PsrfA based promoters was constructed, and a series of mutant PsrfAM promoters with 0.4-1.5-fold strength of native PsrfA were selected. Subsequently, the ComQXPA-PsrfAM QS circuit was utilized to upregulate the expression of red fluorescent protein, and the same QS-based hfq-sRNA system was utilized to downregulate the expression of GFP simultaneously. Last, this bifunctional ComQXPA-PsrfAM QS circuit was verified again by fine-tuning the expression of α-amylase. Therefore, the engineered ComQXPA-PsrfAM QS cassette can be applied as a novel bifunctional QS circuit to flexibly control gene expression in C. glutamicum.

Programming adaptive laboratory evolution of 4-hydroxyisoleucine production driven by a lysine biosensor in Corynebacterium glutamicum.

4-Hydroxyisoleucine (4-HIL) is a promising drug for treating diabetes. In our previous study, 4-HIL was synthesized from self-produced L-isoleucine (Ile) in Corynebacterium glutamicum by expressing an Ile dioxygenase gene. Although the 4-HIL production of recombinant strain SZ06 increased significantly, a by-product, L-lysine (Lys) was accumulated because of the share of the first several enzymes in Ile and Lys biosynthetic pathways. In this study, programming adaptive laboratory evolution (ALE) was designed and conducted in SZ06 to promote 4-HIL biosynthesis. At first, a programming evolutionary system pMK was constructed, which contains a Lys biosensor LysG-PlysE and an evolutionary actuator composed of a mutagenesis gene and a fluorescent protein gene. The evolutionary strain SZ06/pMK was then let to be evolved programmatically and spontaneously by sensing Lys concentration. After successive rounds of evolution, nine mutant strains K1 - K9 with significantly increased 4-HIL production and growth performance were obtained. The maximum 4-HIL titer was 152.19 ± 14.60 mM, 28.4% higher than that in SZ06. This titer was higher than those of all the metabolic engineered C. glutamicum strains ever constructed. The whole genome sequencing of the nine evolved strains revealed approximately 30 genetic mutations in each strain. Only one mutation was directly related to the Lys biosynthetic pathway. Therefore, programming ALE driven by Lys biosensor can be used as an effective strategy to increase 4-HIL production in C. glutamicum.

Optimization of ribosomal binding site sequences for gene expression and 4-hydroxyisoleucine biosynthesis in recombinant corynebacterium glutamicum.

4-Hydroxyisoleucine (4-HIL) has potential value for treating diabetes. α-Ketoglutarate (α-KG)-dependent l-isoleucine dioxygenase (IDO) can convert l-isoleucine (Ile) into 4-HIL. In our previous study, 4-HIL was de novo synthesized from glucose by expressing the ido gene in Corynebacterium glutamicum strain SN01, an Ile producer, and neither Ile nor α-KG was added. In this study, ribosomal binding site (RBS) engineering was applied for gene expression and 4-HIL biosynthesis in C. glutamicum. The 18 tested RBS sequences showed greatly differing strengths for expressing ido, and 8.10-104.22 mM 4-HIL was produced. To supply the cosubstrate α-KG at different levels, the odhI gene was then expressed using the RBS sequences of high, medium, and low strength in the above mentioned optimal strain SF01 carrying R8-ido. However, 4-HIL production decreased to varying amounts, and in some strains, the α-KG was redirected into l-glutamate synthesis. Next, the O2 supply was further enhanced in three ido-odhI coexpressing strains by overexpressing the vgb gene, and 4-HIL production changed dramatically. 4-HIL (up to 119.27 ± 5.03 mM) was produced in the best strain, SF08, suggesting that the synchronic supply of cosubstrates α-KG and O2 is critical for the high-yield production of 4-HIL. Finally, the avtA gene and the ldhA-pyk2 cluster were deleted separately in SF08 to reduce pyruvate-derived byproducts, and 4-HIL production increased to 122.16 ± 5.18 and 139.82 ± 1.56 mM, respectively, indicating that both strains were promising candidates for producing 4-HIL. Therefore, fine-tuning ido expression and the cosubstrates supply through RBS engineering is a useful strategy for improving 4-HIL biosynthesis in C. glutamicum.

Dynamic Control of 4-Hydroxyisoleucine Biosynthesis by Modified l-Isoleucine Biosensor in Recombinant Corynebacterium glutamicum.

4-Hydroxyisoleucine (4-HIL), a promising drug for treating diabetes, can be synthesized from the self-produced l-isoleucine (Ile) by expressing the Ile dioxygenase gene ido in Corynebacterium glutamicum. However, the requirement of three substrates, Ile, α-ketoglutarate (α-KG), and O2, makes such de novo biosynthesis difficult to be fulfilled effectively under static engineering conditions. In this study, dynamic control of 4-HIL biosynthesis by the Ile biosensor Lrp-PbrnFE was researched. The native PbrnFE promoter of natural Ile biosensor was still weak even under Ile induction. Through tetA dual genetic selection, several modified stronger PbrnFEN promoters were obtained from the synthetic library of the Ile biosensor. Dynamic regulation of ido expression by modified Ile biosensors increased the 4-HIL titer from 24.7 mM to 28.9-74.4 mM. The best strain ST04 produced even a little more 4-HIL than the static strain SN02 overexpressing ido by the strong PtacM promoter (69.7 mM). Further dynamic modulation of α-KG supply in ST04 by expressing different PbrnFEN-controlled odhI decreased the 4-HIL production but increased the l-glutamate or Ile accumulation. However, synergistic modulation of α-KG supply and O2 supply in ST04 by different combinations of PbrnFEN-odhI and PbrnFEN-vgb improved the 4-HIL production significantly, and the highest titer (135.3 mM) was obtained in ST17 strain regulating all the three genes by PbrnFE7. This titer was higher than those of all the static metabolic engineered C. glutamicum strains ever constructed. Therefore, dynamic regulation by modified Ile biosensor is a predominant strategy for enhancing 4-HIL de novo biosynthesis in C. glutamicum.