Structure-Function Relationships in Bacterial Regulatory Proteins and an Enzyme Involved in Antibiotic Biosynthesis

The first part of my thesis is focused on a new family of two-component response regulator proteins: Aspartate-Less Regulators (ALR). They lack the catalytic aspartate residue required for the phosphorylation mechanism of typical two component response regulators. We are using biophysical tools to characterize two proteins with redox-sensitive ALR domains: repressor of iron transport regulator (RitR) from Streptococcus pneumonia R6 and diguanylate cyclase Q15Z91 from Pseudoalteromonas atalantica. The structure of inactive RitRC128S monomer showed that the ALR domain and the DNA-binding domain are linked by an α-helix that runs the length of the entire protein, with C128 near the C-terminal end. Bioinformatic analysis of all streptococcal RitR homologs showed that Cys128 is strictly conserved, suggesting that RitR may be a novel redox sensor. Hydrogen peroxide was used to oxidize the cysteine thiol group to determine the structure of the oxidized, dimeric form of RitR. Oxidation of C128 to the disulfide caused a conformational change that caused the DBD to release from the ALR domain. Surprisingly, the freed DBD was observed bound to the ALR domain of the other, disulfide-linked molecule of RitR, recapitulating almost exactly the structure of the inactive, monomeric protein. An extended dimeric conformation was found in the RitRL86A/V93A variant. It binds to the target DNA according to gel filtration and differential scanning fluorimetry. The crystal structure of the RitRL86A/V93A ALR domain showed an unprecedented conformational change for a response regulator protein, where helix α4 is disordered and the two protomers swap their α5 helices to form the dimer. Combined with the C128D mutant in vivo studies, it seems that oxidation of C128 is part of the activation mechanism, but there must be an additional factor that leads to dimerization of the ALR domains. The second ALR protein Q15Z91 has R61 replacing the phosphorylatable aspartate residue in the ALR domain. According to the structure of Q15Z91 with GTP and c-di-GMP, purified Q15Z91 is an activated but product-inhibited dimer. C142 is conserved in the same position as C128 in RitR, and substitution demonstrated that C142 residue is also a redox sensor that involved in Q15Z91 activity regulation. The second part is a mechanistic enzymology project aimed at understanding the structure and mechanism of the novel pyridoxal-5’-phosphate (PLP)-dependent L-arginine hydroxylase/deaminase, MppP, from Streptomyces wadayamensis (SwMppP). SwMppP is predicted to be a type I/II aminotransferase based on primary sequence identity. However, NMR and ESI-MS results showed that SwMppP is not an aminotransferase, but rather a hydroxylase. The enzyme catalyzes the oxygen-dependent hydroxylation of L-arginine, forming 4-hydroxy-2-ketoarginine and the abortive side-product 2-ketoargine in a ratio of 1.7:1. This is exciting because SwMppP is the first PLP-dependent enzyme to react with oxygen in any context other than oxidative decarboxylation. The discovery of this new activity is especially surprising given that the tertiary structure of SwMppP is very similar to that of the prototypical aminotransferase, the E. coli aspartate aminotransferase (PDB entry 1ARS; RMSD of aligned Cα atoms is 3.7 Å). The major differences between the two enzymes are the disordered N terminus of SwMppP, and changes of a limited number of amino acids near the PLP cofactor. The N-terminal helix transitions from a disordered, random-coil state to a helical conformation covering the active site only if/when the substrate is bound. Specific roles of the un-conserved residues in the active site are being studied by mutagenesis. So far, most of the SwMppP mutants have lost the hydroxylase activity and only produce abortive side product 2-ketoarginine. Our mechanistic studies have revealed that formation of the fully oxidized (hydroxylated) product requires 2 equivalents of dioxygen, while formation of 2-ketoarginine requires only one equivalent of dioxygen. Interestingly, the hydroxyl group of 4-hydroxy-2-ketoarginine comes from H2O, not dioxygen. Mutagenesis, structural and kinetic studies were used to understand how the residues in the active site stabilize the quinonoid form of the L-arginine-PLP complex to promote the reaction with dioxygen. Our structural and kinetic characterization of the wild-type and variant forms of SwMppP have allowed us to propose a model where the oxygen incorporated in the hydroxy-arginine product is derived from water rather than from dioxygen. In addition, SwMppP exhibits very high substrate specificity. Either change on the substrate length or guanidine group would result in no binding affinity or little activity

Heterologous protein display on the cell surface of lactic acid bacteria mediated by the s-layer protein

<p>Abstract</p> <p>Background</p> <p>Previous studies have revealed that the C-terminal region of the S-layer protein from <it>Lactobacillus </it>is responsible for the cell wall anchoring, which provide an approach for targeting heterologous proteins to the cell wall of lactic acid bacteria (LAB). In this study, we developed a new surface display system in lactic acid bacteria with the C-terminal region of S-layer protein SlpB of <it>Lactobacillus crispatus </it>K2-4-3 isolated from chicken intestine.</p> <p>Results</p> <p>Multiple sequence alignment revealed that the C-terminal region (LcsB) of <it>Lb. crispatus </it>K2-4-3 SlpB had a high similarity with the cell wall binding domains S_Aand CbsA of <it>Lactobacillus acidophilus </it>and <it>Lb. crispatus</it>. To evaluate the potential application as an anchoring protein, the green fluorescent protein (GFP) or beta-galactosidase (Gal) was fused to the N-terminus of the LcsB region, and the fused proteins were successfully produced in <it>Escherichia coli</it>, respectively. After mixing them with the non-genetically modified lactic acid bacteria cells, the fused GFP-LcsB and Gal-LcsB were functionally associated with the cell surface of various lactic acid bacteria tested. In addition, the binding capacity could be improved by SDS pretreatment. Moreover, both of the fused proteins could simultaneously bind to the surface of a single cell. Furthermore, when the fused DNA fragment of <it>gfp:lcsB </it>was inserted into the <it>Lactococcus lactis </it>expression vector pSec:Leiss:Nuc, the GFP could not be secreted into the medium under the control of the <it>nisA </it>promoter. Western blot, in-gel fluorescence assay, immunofluorescence microscopy and SDS sensitivity analysis confirmed that the GFP was successfully expressed onto the cell surface of <it>L. lactis </it>with the aid of the LcsB anchor.</p> <p>Conclusion</p> <p>The LcsB region can be used as a functional scaffold to target the heterologous proteins to the cell surfaces of lactic acid bacteria <it>in vitro </it>and <it>in vivo</it>, and has also the potential for biotechnological application.</p

Industry-finance integration activities is beneficial to the improvement of business performance of fishery companies—Based on DEA-CCR Model and DEA-Malmquist Model

Fisheries are an essential component of the national economy. The evaluation of fishery enterprises' business performance helps reveal the fishery industry's operating efficiency and serve the fishery economy's sustainable and high-quality development. This study used the data of fishery companies with industry-finance integration on the stock market from 2012-2021 and conducted a comparative study on the business performance of fishery companies with industry-finance integration on the stock market based on the DEA-CCR model and DEA- Malmquist index method. Two main results were revealed (i) the integration of industry and finance was an effective means to improve business performance, and the financial gains from the integration of industry and finance by listed fishery companies improve the overall efficiency and total factor productivity (TFP) of enterprises. (ii) The average TFP of listed fishery companies in China is declining, and technological decline is the main reason for the decline in TFP

Economic analysis of wind power consumption promoted by regenerative electric heating with high proportion of renewable energy

With the rapid development of wind power, the randomness and volatility of wind power have also caused severe problems of wind power consumption. The phenomenon of wind abandonment is particularly prominent in the "three north" areas. The environmental protection and controllability of regenerative electric heating provide a way for wind absorption and abandonment. Therefore, this paper proposes a model to promote wind power consumption by using regenerative electric heating. Firstly, the principle and advantages of the heat storage electric heating equipment are described; secondly, the fine modeling of regenerative electric heating is carried out;then, the mode of using regenerative electric heating to promote wind power consumption is designed;finally, an example is given to analyze the wind power consumption effect and the revenue of load aggregators.Therefore, it is verified that this mode can effectively promote wind power consumption and reduce wind abandon generation, and provide reference for alleviating wind abandon and power limit problem

RitR is an archetype for a novel family of redox sensors in the streptococci that has evolved from two-component response regulators and is required for pneumococcal colonization

To survive diverse host environments, the human pathogen Streptococcus pneumoniae must prevent its self-produced, extremely high levels of peroxide from reacting with intracellular iron. However, the regulatory mechanism(s) by which the pneumococcus accomplishes this balance remains largely enigmatic, as this pathogen and other related streptococci lack all known redox-sensing transcription factors. Here we describe a two-component-derived response regulator, RitR, as the archetype for a novel family of redox sensors in a subset of streptococcal species. We show that RitR works to both repress iron transport and enable nasopharyngeal colonization through a mechanism that exploits a single cysteine (Cys128) redox switch located within its linker domain. Biochemical experiments and phylogenetics reveal that RitR has diverged from the canonical two-component virulence regulator CovR to instead dimerize and bind DNA only upon Cys128 oxidation in air-rich environments. Atomic structures show that Cys128 oxidation initiates a "helical unravelling" of the RitR linker region, suggesting a mechanism by which the DNA-binding domain is then released to interact with its cognate regulatory DNA. Expanded computational studies indicate this mechanism could be shared by many microbial species outside the streptococcus genus

Study on Formulation, in vivo Exposure, and Passive Targeting of Intravenous Itraconazole Nanosuspensions

The pharmacokinetic profile of a drug can be different when delivered as a nanosuspension compared with a true solution, which may in turn affect the therapeutic effect of the drug. The goal of this study was to prepare itraconazole nanosuspensions (ITZ-Nanos) stabilized by an amphipathic polymer, polyethylene glycol-poly (benzyl aspartic acid ester) (PEG-PBLA), by the precipitation-homogenization, and study the pharmacokinetic profile of the ITZ-Nanos. The particle size and morphology of nanosuspensions were determined by Zetasizer and field emission scanning electron microscope (SEM), respectively. The dissolution profile was evaluated using a paddle method according to Chinese Pharmacopoeia 2015. The level of ITZ in plasma and tissues was measured by a HPLC method. The optimized ITZ-Nanos had an average particle size of 268.1 ± 6.5 nm and the particles were in a rectangular form. The dissolution profile of ITZ-Nanos was similar to that of commercial ITZ injections, with nearly 90% ITZ released in the first 5 min. The ITZ-Nanos displayed different pharmacokinetic properties compared with the commercial ITZ injections, including a decreased initial drug concentration, increased plasma half-life and mean residence time (MRT), and increased concentration in the liver, lung, and spleen. The ITZ-Nanos can change the in vivo distribution of ITZ and result in passive targeting to the organs with mononuclear phagocyte systems (MPS)

Masson pine pollen aqueous extract ameliorates cadmium-induced kidney damage in rats

Introduction: Cadmium (Cd) is a hazardous environmental pollutant present in soil, water, and food. Accumulation of Cd in organisms can cause systematic injury and damage to the kidney. The Masson pine pollen aqueous extract (MPPAE) has attracted increasing attention due to its antioxidant activity and ability to enhance immunity.Methods: In this study, we investigated the potential of MPPAE to protect against Cd-induced kidney damage in rats and the underlying mechanism. The transcriptome and metabolome of rats with Cd-induced kidney damage, following treatment with MPPAE, were explored.Results: The concentrations of superoxide dismutase (SOD) and malondialdehyde (MDA) were both significantly altered after treatment with MPPAE. Furthermore, sequencing and analysis of the transcriptome and metabolome of rats with Cd-induced kidney damage, following treatment with MPPAE, revealed differential expression of numerous genes and metabolites compared with the untreated control rats. These differentially expressed genes (DEGs) included detoxification-related genes such as cytochrome P450 and the transporter. The differentially expressed metabolites (DEMs) included 4-hydroxybenzoic acid, L-ascorbate, and ciliatine. Conjoint transcriptome and metabolome analysis showed that several DEGs were correlated with DEMs.Conclusion: These preliminary findings indicate the potential of MPPAE for the treatment of toxic metal poisoning

Design, synthesis, cytotoxic activity and molecular docking studies of new 20(S)-sulfonylamidine camptothecin derivatives

20(S)-Sulfonylamidine CPT-derivatives were prepared and tested for cytotoxicity.Several analogs showed superior cytotoxic activity compared to irinotecan.Key structural features related to cytotoxicity were identified by SAR analysis.Compounds 9 and 15c interacted with Topo I-DNA by a different binding mode from CPT.These compounds are new generation CPT-derived antitumor agents.In an ongoing investigation of 20-sulfonylamidine derivatives (9, YQL-9a) of camptothecin (1) as potential anticancer agents directly and selectively inhibiting topoisomerase (Topo) I, the sulfonylamidine pharmacophore was held constant, and a camptothecin derivatives with various substitution patterns were synthesized. The new compounds were evaluated for antiproliferative activity against three human tumor cell lines, A-549, KB, and multidrug resistant (MDR) KB subline (KBvin). Several analogs showed comparable or superior antiproliferative activity compared to the clinically prescribed 1 and irinotecan (3). Significantly, the 20-sulfonylamidine derivatives exhibited comparable cytotoxicity against KBvin, while 1 and 3 were less active against this cell line. Among them, compound 15c displayed much better cytotoxic activity than the controls 1, 3, and 9. Novel key structural features related to the antiproliferative activities were identified by structure–activity relationship (SAR) analysis. In a molecular docking model, compounds 9 and 15c interacted with Topo I-DNA through a different binding mode from 1 and 3. The sulfonylamidine side chains of 9 and 15c could likely form direct hydrogen bonds with Topo I, while hydrophobic interaction with Topo I and π–π stacking with double strand DNA were also confirmed as binding driving forces. The results from docking models were consistent with the SAR conclusions. The introduction of bulky substituents at the 20-position contributed to the altered binding mode of the compound by allowing them to form new interactions with Topo I residues. The information obtained in this study will be helpful for the design of new derivatives of 1 with most promising anticancer activity.CPT (green), 9 (magenta), and 15c (blue) in the binding site of DNA-Topo-I

Efficacy and safety of XELOX combined with anlotinib and penpulimab vs XELOX as an adjuvant therapy for ctDNA-positive gastric and gastroesophageal junction adenocarcinoma: a protocol for a randomized, controlled, multicenter phase II clinical trial (EXPLORING study)

BackgroundThe efficacy of current adjuvant chemotherapy for gastric adenocarcinoma/gastroesophageal junction adenocarcinoma (GA/GEJA) leaves much to be desired. ctDNA could serve as a potential marker to identify patients who are at higher risk of recurrence. Reinforcing standard adjuvant chemotherapy with immunotherapy has already been indicated to significantly improve clinical outcome, albeit such evidence is rare in GA/GEJA. Here, we intend to explore the clinical benefit of the reinforcement of adjuvant immunotherapy and antiangiogenics alongside with chemotherapy in patients who are deemed in high risk of recurrence by ctDNA analysis, which might shed light on further improvements in adjuvant therapy for GA/GEJA.Methods/DesignThis study is designed as a prospective, multicenter, randomized, controlled phase II study in patients histologically or cytologically diagnosed with GA/GEJA who underwent D2 gastrectomy and achieved R0 or R1 resection. From February 2022, a total of 300 stage III patients will be enrolled and subjected according to ctDNA sequencing results, and those with positive results will subsequently be randomized 1:1 to arm A or B. Patients in arm A will receive anlotinib, penpulimab and XELOX for 6-8 cycles, maintained with anlotinib and penpulimab for up to 1 year, while patients in arm B will receive XELOX alone for 6-8 cycles. ctDNA-negative patients will be assigned to arm C, and patients who are ctDNA positive but failed in randomization will be assigned to arm D. Patients in arms C and D will receive the investigator’s choice of therapy. The primary endpoint is the median disease-free survival (DFS) of arm A versus arm B determined via CT/MRI imaging. Secondary endpoints include the DFS of ctDNA positive patients versus ctDNA negative patients, the 2- and 3-year DFS rates, overall survival (OS), the impact of hallmark molecules on the treatment response, adverse events (AEs), and the impact of nutrition status or exercise on recurrence.DiscussionWe expect that ctDNA would be a strong prognostic factor and ctDNA-positive patients are at higher risk of relapse than ctDNA-negative patients. The addition of anlotinib and penpulimab to XELOX, may contribute to delaying relapse in ctDNA-positive patients.Trial registrationhttps://www.clinicaltrials.gov, identifier NCT05494060

Default risk, state ownership and the cross-section of stock returns: evidence from China

We apply a structural model to estimate firm-level default risk in China and investigate the stock return predictability of default risk and the moderating effects of state ownership for the sample period from 2003 to 2015. We show unique evidence that in China, default risk is positively associated with expected stock returns and state ownership matters considerably to the return predictability of default risk. We find investors of state-owned enterprises (SOEs) are not compensated appropriately in China despite of their higher default risk exposure. Our empirical evidence supports the conjecture on shareholder advantages and suggests that a strong bargaining power of equity holders would have a negative impact on stock returns