MvaT binds to the PexsC promoter to repress the type III secretion system in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen capable of causing a variety of acute and chronic infections. Its type III secretion system (T3SS) plays a critical role in pathogenesis during acute infection. ExsA is a master regulator that activates the expression of all T3SS genes. Transcription of exsA is driven by two distinct promoters, its own promoter PexsA and its operon promoter PexsC. Here, in combination with a DNA pull-down assay and mass spectrometric analysis, we found that a histone-like nucleoid-structuring (H-NS) family protein MvaT can bind to the PexsC promoter. Using EMSA and reporter assays, we further found that MvaT directly binds to the PexsC promoter to repress the expression of T3SS genes. The repression of MvaT on PexsC is independent of ExsA, with MvaT binding to the -429 to -380 bp region relative to the transcription start site of the exsC gene. The presented work further reveals the complex regulatory network of the T3SS in P. aeruginosa.

Localizing the position of the Segond fracture bed under CT measurements to determine the functional tibial insertion of an anterolateral ligament.

Background: Many studies have confirmed the existence of ligament structures in the anterolateral region of the knee that maintain rotational stability of the knee joint, namely, the anterolateral ligament (ALL). Most scholars believe that knee joint reconstruction should be considered during revision surgery and a high level of pivot displacement test (stage 2 or 3). During ALL reconstruction, the choice of ligament reconstruction sites affects the success rate and prognosis of the operation. Therefore, the choice of ligament reconstruction sites is particularly important. There is little research on the lateral ALL tibia insertion point, and most clinicians use the midpoint Gerdy's tubercle and fibular head as insertion points. However, the reconstruction effect is not ideal. Objective: This study aims to measure the position of the Segond fracture bed on CT images to determine the ALL position of the tibia. Method: To determine the position of the Segond fracture bone bed, the CT AM Volume Share 2 system was used to manually measure the position of bone fragments in 23 Segond fracture patients. Using the highest point of Gerdy's tubercle in the CT axial slices and the outermost point of the fibular head in the CT axial slices as reference points, the direction and angle of the CT slices were adjusted to ensure that the highest point of the Gerdy tubercle, the outermost point of the fibular head, and the center of Segond fracture bed were in the same sagittal slice. A CT sagittal slice measures the vertical distance from the center of the Segond fracture bed to the Gerdy-fibular line segment (G-F line segment), which is the line connecting the highest point of the segment to the outermost point of the fibula. The distance from the vertical point at the center of the Segond fracture bed of the G-F line to the highest point of the Gerdy tubercle was measured. All measurements were performed using the same measurement standard and were expressed as a percentage of the length of the G-F line. The measured results were statistically analyzed using SPSS 25.0 descriptive statistical research methods. Results: The average length of the G-F segment measured on CT images was 39.6 ± 2.0 mm, and the average vertical length from the center of the Segond fracture bed to the G-F segment was 13.1 ± 1.1 mm, accounting for 33.2% ± 2.1% of the length of the G-F segment. The length from the vertical point of the fracture bed on the G-F line segment to the highest point of the Gerdy tubercle was 14.7 ± 1.3 mm, accounting for 37.1% ± 2.9% of the length of the G-F segment. Conclusion: Through the study of the CT measurement of the Segond fracture location, we obtained the location of the functional tibial insertion of ALL, which is different from the anatomical insertion of ALL and is more inclined to the Gerdy tubercle and above, which has reference value for the treatment of recovering the function of anterolateral ligament after reconstruction.

MvfR Controls Tolerance to Polymyxin B by Regulating rfaD in Pseudomonas aeruginosa.

Polymyxins are currently the last-resort antibiotics for the treatment of multidrug-resistant Gram-negative bacterial infections. To expand the understanding of the intrinsic resistance mechanism against polymyxins, a laboratory strain of Pseudomonas aeruginosa PAO1 was subjected to serial passage in the presence of sublethal doses of polymyxin B over a period of 30 days. By whole-genome sequencing of successively isolated polymyxin B-resistant isolates, we identified a frameshift mutation (L183fs) in the mvfR gene that further increased polymyxin resistance in the pmrB mutant background. A ΔmvfR mutation alone showed higher tolerance to polymyxin B due to altered lipopolysaccharide (LPS) on the surface of bacterial cells, which decreases its outer membrane permeability. In the ΔmvfR mutant, polymyxin B treatment caused the upregulation of rfaD, the gene involved in LPS core oligosaccharide synthesis, which is responsible for polymyxin tolerance. To the best of our knowledge, this is the first report of mvfR mutation conferring polymyxin resistance in P. aeruginosa via increased integrity of bacterial outer membrane. IMPORTANCE Antibiotic resistance imposes a considerable challenge for the treatment of P. aeruginosa infections. Polymyxins are the last-resort antibiotics for the treatment of multidrug-resistant P. aeruginosa infections. Understanding the development and mechanisms of bacterial resistance to polymyxins may provide clues for the development of new or improved therapeutic strategies effective against P. aeruginosa. In this study, using an in vitro evolution assay in combination with whole-genome sequencing, we demonstrated that MvfR controls tolerance to polymyxin B by regulating the rfaD gene in P. aeruginosa. Our results reveal a novel mechanism employed by P. aeruginosa in the defense against polymyxin antibiotics.

1,25-Dihydroxyvitamin D regulates macrophage activation through FBP1/PKR and ameliorates arthritis in TNF-transgenic mice.

1,25-Dihydroxyvitamin D (1,25(OH)2D3) has immunomodulatory activity and its deficiency correlates with rheumatoid arthritis (RA) incidence. Whether 1,25(OH)2D3 modulates macrophage activation or protects against RA remains unclear. We demonstrate that 1,25(OH)2D3 suppresses M1 macrophage polarization and CD80, IL-6, CXCL10, IFIT1, IFI44, and double-stranded RNA-dependent protein kinase R (PKR) expression in the macrophages of RA patients. In phorbol 12-myristate 13-acetate-induced THP-1 cells, 1,25(OH)2D3 upregulates fructose-1,6-bisphosphatase 1 (FBP1) expression through direct promoter interaction. FBP1 interacts with PKR and promotes PKR ubiquitination degradation. SiR-FBP1 transfection impairs 1,25(OH)2D3 action and suppresses IL-6, CXCL10, IFIT1, IFI27, and IFI44 expression in macrophages, whereas siR-PKR transfection impairs siR-FBP1 activity in 1,25(OH)2D3-treated macrophages. 1,25(OH)2D3 treatment ameliorates the clinical signs of arthritis in tumor necrosis factor-transgenic mice, inhibits M1 polarization and marker expression, and promotes FBP1 expression in mononuclear cells isolated from swollen joints; thus, 1,25(OH)2D3 suppresses M1 macrophage activation through FBP1/PKR and ameliorates arthritis by restoring the macrophage subtype.

Whole exome sequencing of lung adenocarcinoma and lung squamous cell carcinoma in one individual: A case report.

Multiple primary lung cancers (MPLCs) refers to two or more primary malignant tumors that occur simultaneously or successively in the lung of the same patient. Distinguishing MPLCs from intrapulmonary metastases is important for treatment strategy and prognosis. MPLCs have been considered as having different origins and clonal evolutionary processes. Whole genome sequencing (WGS) and whole exome sequencing (WES) are regarded as an effective way to identify the relationship and differentiation among MPLC nodules. Here, we report the case of a 63-year-old MPLC male patient who smoked for 40 years. Two nodules were found on chest computed tomography (CT) scan, which were further confirmed by pathology to be lung adenocarcinoma (ADC) and lung squamous cell carcinoma (SCC), respectively. WES of the two different nodules was performed, and the results showed that there was a significant genetic difference between the two nodules. Further analysis of the tumor mutation burden (TMB) of the two tumor lesions showed that the TMB of the squamous cell carcinoma was higher than that of the adenocarcinoma, indicating that the squamous cell carcinoma had a higher mutation frequency. According to the pathology and WES sequencing results, MPLCs for this case were regarded as independent of each other, with different origins and clonal evolutionary processes. In this case report, we emphasize that WES should play an important role in determining the origin of MPLC clones, and also make some explorations for the further discovery of new potential driver genes and therapeutic targets.