Interactions between plants and bacterial communities for phytoremediation of petroleum-contaminated soil.

Plants can stimulate the microbes to degrade ubiquitous petroleum hydrocarbons (PHCs), which has prompted a novel view on rhizoremediation. In the present study, the degradation rate of PHCs was investigated and 16S rRNA gene analysis was performed to investigate the PHC-degrading bacteria in petroleum-contaminated soil with different plants. Mirabilis jalapa (M. jalapa) has a higher PHC degradation rate than Lolium perenne (L. perenne) under petroleum contamination. The bacterial diversity in rhizospheric soil was decreased but the relative abundance of Actinobacteriota, Proteobacteria, and Candidatus Saccharibacteria were significant increased on 45 days petroleum-contaminated rhizospheric soil. In addition, the relative expression of PHC degradation-related genes, the content of malic acid and citric acid of the root exudates in the two plants was significantly increased in response to petroleum stress. The content of citric acid increased 11.9 times in M. jalapa and 3.4 times in L. perenne, respectively, in response to petroleum stress. These results indicate that M. jalapa changes the hydrocarbon-degrading microbial community to enhance the degradation of PHCs by root exudates and phytostimulation.

Cognition, affect, and behavioural changes among university students after 10 new guidelines were issued in China: An exploratory study.

Objectives: Since late 2019, the coronavirus disease 2019 (COVID-19) epidemic has become a common public health concern globally. China has entered a new phase of prevention and control with the implementation of the 10 new guidelines epidemic prevention policy in early December 2022. The resurgence of the outbreak may cause negative consequences on the behaviour of university students. This study aimed to assess the relationship between cognition, affect, and behavioural changes among university students and the related influencing factors after 10 new guidelines were issued, as well as the difficulties or concerns encountered in the current epidemic prevention process. It also provides a reference for the government to formulate targeted epidemic prevention strategies. Methods: This study is a cross-sectional investigation. Self-designed questionnaires were distributed to students of a university in Hangzhou between December 25, 2022, and March 13, 2023, using convenience and snowball sampling methods for online surveys. Data analysis involved descriptive analysis, non-parametric tests, correlation, multiple linear regression, and content analyses. Results: University students had a moderate to high level of cognition about COVID-19 and a medium level of affect. However, the level of behavioural changes was low and the average score was 2.33 (2.00, 3.00). Multiple linear regression analysis revealed that female sex, higher grade, medical specialty, affective factor, and cognitive factor were influencing factors of behavioural changes, which accounted for 35.7% of the variance in behavioural change. Difficulties or concerns included apprehension (84.8%), lack of information (39.3%), and uncertainty about the future (55.1%). Conclusions: The prevention behaviour of university students has slackened. Evidence-based tailored policy development is indicated. This study suggested that schools and the government can improve the effectiveness of epidemic prevention among university students by adjusting the strategy of epidemic prevention policy formulation, broadening the channels of epidemic prevention information dissemination, and improving the mechanism of "government-community-school-family" collaborative governance.

Chemical reagents for the enrichment of modified peptides in MS-based identification.

Chemical reagents with special groups as enrichable handles have empowered the ability to label and enrich modified peptides. Here is an overview of different chemical reagents with affinity tags to isolate labeled peptides and the latest developments of enrichment strategies. Biotin is the most used affinity tag due to its high interaction with avidin. To decrease the unfavorable influence of biotin for its poor efficiency in ionization and fragmentation in downstream MS analysis, cleavable moieties were installed between the reactive groups and biotin to release labeled peptides from the biotin. To minimize the steric hindrance of biotin, a two-step method was developed, for which alkyne- or azide-tagged linkers were firstly used to label peptides and then biotin was installed through click chemistry. Recently, new linkers using a small phosphonic acid as the affinity tag for IMAC or TiO2 enrichment have been developed and successfully used to isolate chemically labeled peptides in XL-MS. A stable P-C instead of P-O bond was introduced to linkers to differentiate labeled and endogenous phosphopeptides. Furthermore, a membrane-permeable phosphonate-containing reagent was reported, which facilitated the study of living systems. Taking a cue from classic chemical reactions, stable metal-complex intermediates, including cobalt and palladium complexes, have been developed as peptide purification systems. Advanced enrichment strategies have also been proposed, such as the two-stage IMAC enrichment method and biotin-based two-step reaction strategy, allowing the reduction of unwanted peptides and improvements for the analysis of specific labeled peptides. Finally, future trends in the area are briefly discussed.

A strategy to enhance and modify fatty acid synthesis in Corynebacterium glutamicum and Escherichia coli: overexpression of acyl-CoA thioesterases.

BACKGROUND: Fatty acid (FA) is an important platform compound for the further synthesis of high-value biofuels and oleochemicals, but chemical synthesis of FA has many limitations. One way to meet the future demand for FA could be to use microbial cell factories for FA biosynthesis. RESULTS: Thioesterase (TE; TesA, TesB, and TE9) of Corynebacterium glutamicum (CG) can potentially improve FA biosynthesis, and tesA, tesB, and te9 were overexpressed in C. glutamicum and Escherichia coli (EC), respectively, in this study. The results showed that the total fatty acid (TFA) production of CGtesB and ECtesB significantly increased to 180.52 mg/g dry cell weight (DCW) and 123.52 mg/g DCW, respectively (P < 0.05). Overexpression strains CG and EC could increase the production of C16:0, C18:1(t), C18:2, C20:1, C16:1, C18:0, and C18:1(c) (P < 0.05), respectively, and the changes of long-chain FA resulted in the enhancement of TFA production. The enzymatic properties of TesA, TesB, and TE9 in vitro were determined: they were specific for long-, broad and short-chain substrates, respectively; the optimal temperature was 30.0 °C and the optimal acid-base (pH) were 8.0, 8.0, and 9.0, respectively; they were inhibited by Fe2+, Cu2+, Zn2+, Mg2+, and K+. CONCLUSION: Overexpression TE enhances and modifies FA biosynthesis with multiple productive applications, and the enzyme properties provided useful clues for optimizing FA synthesis.

Application of a Chitosan-based Active Packaging Film Prepared with Cell-free Supernatant of Lacticaseibacillus paracasei ALAC-4 in Mongolian Cheese Preservation.

Fungal spoilage of food is a worldwide concern prompting the development of many antimicrobial agents and applications. In this study, the cell-free supernatant (CFS) of Lacticaseibacillus paracasei ALAC-4 had a significant inhibition effect on fungi. The CFS with antifungal activities were combined with chitosan (CS) matrix to prepare an active packaging CS-CFS films by using a solvent casting method and used for the packaging of Mongolian cheese for 15 days during storage at 4 ± 1℃. The optimized formulation of the film were 1.25% (w/v) chitosan, 1.75% (w/v) gelatin, 0.3% (v/v) glycerol, and 9.6% (w/v) CFS. It was found that CS-CFS films exhibited strong antifungal activities against molds and yeasts, especially Candida albicans, and also had excellent mechanical properties. Additionally, FTIR spectroscopy indicated that hydrogen bonds between the CFS and CS formed, and there was a smooth surface, compact cross-section observed in SEM morphologies of CS-CFS films. Furthermore, CS-CFS film also displayed a strong antifungal effect against molds and yeasts on cheese surface. These results suggest that the chitosan-based CS-CFS film has a promising application for Mongolian cheese and food preservation.

Development of a MMAE-based antibody-drug conjugate targeting B7-H3 for glioblastoma.

B7-H3 (immunoregulatory protein B7-homologue 3) is overexpressed in many cancer cells with limited expression in normal tissues, considered to be a promising target for tumor therapeutics. Clinical trials of antibody-drug conjugates (ADCs) against different targets for glioblastoma have been investigated and showed potent efficacies. In this study, we developed a homogeneous ADC 401-4 with a drug-to-antibody ratio (DAR) of 4, which was prepared by conjugation of Monomethyl auristatin E (MMAE) to a humanized anti-B7-H3 mAb 401, through a divinylsulfonamide-mediated disulfide re-bridging approach. In vitro studies, 401-4 displayed specific killing against B7-H3-expressing tumors and was more effective in cells with higher levels of B7-H3 for different glioblastoma cells. 401-4 was furthered labeled with Cy5.5 to yield a fluorescent conjugate 401-4-Cy5.5. The in vivo imaging studies showed that the conjugate accumulated in tumor regions and exhibited the ability to target-specific delivery. In addition, significant antitumor activities for 401-4 was observed against U87-derived tumor xenografts in a dose dependent manner.

Isolation, characterization and application of bacteriophage PSDA-2 against Salmonella Typhimurium in chilled mutton.

Salmonella is a common foodborne pathogen, especially in meat and meat products. Lytic phages are promising alternatives to conventional methods for Salmonella biocontrol in food and food processing. In this study, a virulent bacteriophage (PSDA-2) against Salmonella enterica serovar Typhimurium was isolated from the sewage and it was found that PSDA-2 belongs to Cornellvirus genus of Siphoviridae family by morphological and phylogenetic analysis. Based on the one-step growth curve, PSDA-2 has a short latent period (10 min) and a high burst size (120 PFU/cell). The stability test in vitro reveals that PSDA-2 is stable at 30-70°C and pH 3-10. Bioinformatics analysis show that PSDA-2 genome consists of 40,062 bp with a GC content of 50.21% and encodes 63 open reading frames (ORFs); no tRNA genes, lysogenic genes, drug resistance genes and virulence genes were identified in the genome. Moreover, the capacity for PSDA-2 to control Salmonella Typhimurium in chilled mutton was investigated. The results show that incubation of PSDA-2 at 4°C reduced recoverable Salmonella by 1.7 log CFU/mL and 2.1 log CFU/mL at multiplicity of infection (MOI) of 100 and 10,000 respectively, as relative to the phage-excluded control. The features of phage PSDA-2 suggest that it has the potential to be an agent to control Salmonella.

Hepatocyte-specific TAK1 deficiency drives RIPK1 kinase-dependent inflammation to promote liver fibrosis and hepatocellular carcinoma.

Transforming growth factor ß-activated kinase1 (TAK1) encoded by the gene MAP3K7 regulates multiple important downstream effectors involved in immune response, cell death, and carcinogenesis. Hepatocyte-specific deletion of TAK1 in Tak1ΔHEP mice promotes liver fibrosis and hepatocellular carcinoma (HCC) formation. Here, we report that genetic inactivation of RIPK1 kinase using a kinase dead knockin D138N mutation in Tak1ΔHEP mice inhibits the expression of liver tumor biomarkers, liver fibrosis, and HCC formation. Inhibition of RIPK1, however, has no or minimum effect on hepatocyte loss and compensatory proliferation, which are the recognized factors important for liver fibrosis and HCC development. Using single-cell RNA sequencing, we discovered that inhibition of RIPK1 strongly suppresses inflammation induced by hepatocyte-specific loss of TAK1. Activation of RIPK1 promotes the transcription of key proinflammatory cytokines, such as CCL2, and CCR2+ macrophage infiltration. Our study demonstrates the role and mechanism of RIPK1 kinase in promoting inflammation, both cell-autonomously and cell-nonautonomously, in the development of liver fibrosis and HCC, independent of cell death, and compensatory proliferation. We suggest the possibility of inhibiting RIPK1 kinase as a therapeutic strategy for reducing liver fibrosis and HCC development by inhibiting inflammation.

Chronic Stimulation of the Autophagy-inducing Ingredient of Areca Nut Promotes Tumor Growth In Vivo Through Up-regulation of Tumoral Autophagy.

BACKGROUND/AIM: Autophagy can be either tumor promotive or suppressive. We previously identified an autophagy-inducing activity in the 30-100 kDa fraction of areca-nut-extract (ANE 30-100K) and showed that several tumor cells subjected to chronic ANE 30-100K stimulation (CAS) exhibited higher resistance against stressed environments including serum-free (SF) conditions in vitro. Herein, we aimed to assess whether CAS can also provide growth advantages for tumor cells in vivo and the therapeutic effect of autophagy inhibition on CAS-treated tumors. MATERIALS AND METHODS: Esophageal CE81T/VGH cells and nude mice were used as experimental models. Autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ), as well as another anticancer drug cisplatin (DDP), were chosen to challenge CAS-treated CE81T/VGH cells in vitro and in vivo. RESULTS: CAS-treated CE81T/VGH cells expressed higher levels of microtubule-associated protein 1 light chain 3A/B-II (LC3-II) and beclin 1 proteins, and showed stronger resistance to SF and hypoxia conditions, that were mitigated by CQ or 3-MA in vitro. Furthermore, CAS-treated CE81T/VGH cells induced significantly larger tumors in mice, which were also attenuated by single 3-MA or CQ treatment. Finally, the combined treatment of 3-MA or CQ with DDP further up-regulated DDP-induced caspase-3 activity in vitro and exhibited synergistic anti-tumor effects on mice. CONCLUSION: CAS may up-regulate tumoral autophagy and provide growth advantage for tumors both in vitro and in vivo. Furthermore, autophagy inhibition alone or in combination with DDP may achieve positive therapy for tumors encountered with CAS.

Three-dimensional high performance free-standing anode by one-step carbonization of pinecone in microbial fuel cells.

In this paper, the free-standing macroporous carbon anode is prepared by one-step carbonization of pinecone without any further modification. The obtained anode is N, P-codoped porous carbon material, which is beneficial for electrochemical active bacterial adhesion and the fast start-up of cells. Both of the output voltage and long-term operation stability of the obtained anode are higher than that of carbon felt. The charge transfer resistance after biofilm formation is only 1.4 Ω, being 85.1% lower than that of carbon felt anode. 16S rRNA gene sequence analysis shows that Geobacter soli is the main electricigen and its ratio at the obtained anode is much higher than that at carbon felt (77.4% vs 34.0%). The N, P-codoped carbon as the three-dimensional free-standing anode has excellent electrochemical properties and is low cost and easy preparation. Most importantly, it enhances extracellular electron transfer, thus has potential application in microbial fuel cells.

Enhanced 2-keto-L-gulonic acid production by applying L-sorbose-tolerant helper strain in the co-culture system.

2-Keto-L-gulonic acid (the precursor of vitamin C) is bio-converted from L-sorbose by mixed fermentation of Ketogulonicigenium vulgare and a helper strain. The helper strain promotes the conversion of 2-KLG by enhancing the growth of K. vulgare, but its growth is greatly inhibited by high concentration of L-sorbose, which consequently influence the 2-KLG production. The aim of this study is to obtain L-sorbose-tolerant helper strain (LHS) by experimental evolution for reduced L-sorbose-inhibition-effect and enhanced 2-KLG productivity in high concentration of L-sorbose. After three steps screening by using our devised screening strategy, three strains (i.e., Bc 21, Bc 47, Bc 50) with high resistance to high concentration of L-sorbose were obtained. The fermentation tests by co-culturing Bc 21 and K. vulgare 418 showed that the production of 2-KLG was increased by 17.9% in 11% L-sorbose medium than that in 8% after 55 h of fermentation and the conversion rate was 89.5%. The results suggested that Bc 21 could be an ideal helper strain for 2-KLG production under high concentration of L-sorbose and demonstrated the feasibility of using experimental evolution to breed LHS for vitamin C production.

[Roles of astrocytes in cerebral infarction and related therapeutic strategies].

Astrocytes are the most abundant cells in the central nervous system and play significant roles in normal brain. With cerebral infarction, astrocytes are activated as reactive astrocytes and form glial scars, which play an essential part in brain injury. According to their roles in neuroprotection after cerebral infarction, regulation of scar formation, nerve regeneration, maintenance of blood-brain barrier, promotion of angiogenesis and immune response, scholars have proposed a variety of therapeutic strategies based on targeting astrocytes. This article reviews the research progress on the changes in astrocyte signaling pathways before and after cerebral infarction and the related therapeutic strategies.

SuhB is a novel ribosome associated protein that regulates expression of MexXY by modulating ribosome stalling in Pseudomonas aeruginosa.

Translation elongation is modulated by various ribosome-binding proteins. Environmental stresses, such as starvation and antibiotics, can cause stalling of bacterial ribosomes, which may alter gene expression through a transcription or translation attenuation mechanism. In Pseudomonas aeruginosa, the expression of MexXY multidrug efflux system, which plays a significant role in resistance against aminoglycoside antibiotics, is controlled by a translation surveillance mechanism. Stalling of ribosome at the PA5471 leader peptide (PA5471.1) mRNA leads to transcription of PA5471, which subsequently up-regulates the expression of MexXY. In this study, we found that mutation in a suhB gene leads to decreased susceptibility to aminoglycosides. Transcriptomic analysis revealed an up-regulation of MexXY and PA5471, which were demonstrated to be responsible for the decreased susceptibility of the suhB mutant. We further demonstrated that PA5471.1 is essential for the up-regulation of PA5471 in the suhB mutant. Co-immunoprecipitation assay revealed an interaction between SuhB and ribosome, suggesting a role of SuhB in translation. Indeed, higher amount of PA5471.1 mRNA was found to associate with ribosome isolated from the suhB mutant, indicating increased ribosome stalling. Therefore, this study identified SuhB as a novel ribosome associated protein that is involved in modulating ribosome activity.

PrtR homeostasis contributes to Pseudomonas aeruginosa pathogenesis and resistance against ciprofloxacin.

Pseudomonas aeruginosa is an opportunistic pathogen that causes acute and chronic infections in humans. Pyocins are bacteriocins produced by P. aeruginosa that are usually released through lysis of the producer strains. Expression of pyocin genes is negatively regulated by PrtR, which gets cleaved under SOS response, leading to upregulation of pyocin synthetic genes. Previously, we demonstrated that PrtR is required for the expression of type III secretion system (T3SS), which is an important virulence component of P. aeruginosa. In this study, we demonstrate that mutation in prtR results in reduced bacterial colonization in a mouse acute pneumonia model. Examination of bacterial and host cells in the bronchoalveolar lavage fluids from infected mice revealed that expression of PrtR is induced by reactive oxygen species (ROS) released by neutrophils. We further demonstrate that treatment with hydrogen peroxide or ciprofloxacin, known to induce the SOS response and pyocin production, resulted in an elevated PrtR mRNA level. Overexpression of PrtR by a tac promoter repressed the endogenous prtR promoter activity, and electrophoretic mobility shift assay revealed that PrtR binds to its own promoter, suggesting an autorepressive mechanism of regulation. A high level of PrtR expressed from a plasmid resulted in increased T3SS gene expression during infection and higher resistance against ciprofloxacin. Overall, our results suggest that the autorepression of PrtR contributes to the maintenance of a relatively stable level of PrtR, which is permissive to T3SS gene expression in the presence of ROS while increasing bacterial tolerance to stresses, such as ciprofloxacin, by limiting pyocin production.

SuhB is a regulator of multiple virulence genes and essential for pathogenesis of Pseudomonas aeruginosa.

UNLABELLED: During initial colonization and chronic infection, pathogenic bacteria encounter distinct host environments. Adjusting gene expression accordingly is essential for the pathogenesis. Pseudomonas aeruginosa has evolved complicated regulatory networks to regulate different sets of virulence factors to facilitate colonization and persistence. The type III secretion system (T3SS) and motility are associated with acute infections, while biofilm formation and the type VI secretion system (T6SS) are associated with chronic persistence. To identify novel regulatory genes required for pathogenesis, we screened a P. aeruginosa transposon (Tn) insertion library and found suhB to be an essential gene for the T3SS gene expression. The expression of suhB was upregulated in a mouse acute lung infection model, and loss of suhB resulted in avirulence. Suppression of T3SS gene expression in the suhB mutant is linked to a defective translation of the T3SS master regulator, ExsA. Further studies demonstrated that suhB mutation led to the upregulation of GacA and its downstream small RNAs, RsmY and RsmZ, triggering T6SS expression and biofilm formation while inhibiting the T3SS. Our results demonstrate that an in vivo-inducible gene, suhB, reciprocally regulates genes associated with acute and chronic infections and plays an essential role in the pathogenesis of P. aeruginosa. IMPORTANCE: A variety of bacterial pathogens, such as Pseudomonas aeruginosa, cause acute and chronic infections in humans. During infections, pathogens produce different sets of virulence genes for colonization, tissue damage, and dissemination and for countering host immune responses. Complex regulatory networks control the delicate tuning of gene expression in response to host environments to enable the survival and growth of invading pathogens. Here we identified suhB as a critical gene for the regulation of virulence factors in P. aeruginosa. The expression of suhB was upregulated during acute infection in an animal model, and mutation of suhB rendered P. aeruginosa avirulent. Moreover, we demonstrate that SuhB is required for the activation of virulence factors associated with acute infections while suppressing virulence factors associated with chronic infections. Our report provides new insights into the multilayered regulatory network of virulence genes in P. aeruginosa.