The importance of the thickness of femoral lateral wall for treating intertrochanteric fractures: a finite elements analysis.

To explore how the thickness of the femoral lateral wall influences the effectiveness of internal fixation systems used to treat intertrochanteric fractures. CT images of the pelvis and femur of a male adult were used to construct an intertrochanteric fracture model (AO/OTA 31-A2) with various thicknesses of the femoral lateral wall (FLW). Four finite element (FE) models were created with the lateral femoral walls being 10 mm, 20 mm, 30 mm, and 40 mm thick. The fracture models were fixed with a dynamic hip screw (DHS), a proximal femoral nail anti-rotation (PFNA), and a proximal femoral locking compression plate (P-FLCP). A simulated vertical load was applied to the femoral head. The stress and displacement of the implant and femur in each model were recorded for comparison. The FE analysis of the intertrochanteric fracture models showed that the PFNA system could provide better stability than the DHS and P-FLCP with the same thickness of FLW. The FLW provided buttress support to the femoral head and neck when using a DHS and PFNA, and the buttress strength was proportional to the thickness of FLW. The maximum stress in the DHS model was recorded on the DHS plate which accommodated the lag screw. For the PFNA model, the maximum stress appeared at the connection between the nail and blade. In the P-FLCP model, the maximum stresses were highly concentrated at the connection between the cephalic nails and the proximal plate. The thickness of the femoral lateral wall should be considered an important factor when selecting a suitable internal fixation system for intertrochanteric fractures. Based on the FE analysis, intramedullary fixation, such as PFNA, experiences lower stress levels and a moderate displacement in comparison to DHS and P-FCLP when used to treat intertrochanteric fractures.

Characterization and engineering control of the effects of reactive oxygen species on the conversion of sterols to steroid synthons in Mycobacterium neoaurum.

The conversion of sterols to steroid synthons by engineered mycobacteria comprises one of the basic ways for the production of steroid medications in the pharmaceutical industry. Here, we revealed that high amounts of reactive oxygen species (ROS) generate during the conversion process of sterols, which impairs the cell viability of mycobacterial cells and thus hinders the conversion of sterols to steroid synthons. Accordingly, the endogenous antioxidants for detoxifying ROS in mycobacteria, ROS scavenging enzymes and low molecular weight thiols, were examined. The results revealed that three antioxidants, catalase (CAT), mycothiol (MSH), and ergothioneine (EGT), demonstrated efficacy toward neutralizing the excessive ROS produced during sterol metabolism. CAT overexpression or MSH or EGT augmentation enhanced the conversion of phytosterols to 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) by 18.9%, 23.8%, and 32.1%, respectively, and also enhanced the cell viability, indicating the benefits of these antioxidants in reducing ROS-induced stress. Further combinatorial augmentation of CAT, MSH, and EGT demonstrated enhanced effects toward intracellular ROS scavenging, resulting in 54.2% greater cell viability and 47.5% enhancement in 4-HBC production. These findings indicated that the excessive ROS induces cell stress, in turn limiting the conversion of sterols, whereas neutralization of the excessive ROS by combined control of CAT, MSH, and EGT serves as an effective strategy to boost the conversion productivity of sterols to steroid synthons.

Enhanced conversion of sterols to steroid synthons by augmenting the peptidoglycan synthesis gene pbpB in Mycobacterium neoaurum.

Some species of mycobacteria have been modified to transform sterols to valuable steroid synthons. The unique cell wall of mycobacteria has been recognized as an important organelle to absorb sterols. Some cell wall inhibitors (e.g., vancomycin and glycine) have been validated to enhance sterol conversion by interfering with transpeptidation in peptidoglycan biosynthesis. Therefore, two transpeptidase genes, pbpA and pbpB, were selected to rationally modify the cell wall to simulate the enhancement effect of vancomycin and glycine on sterol conversion in a 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) producing strain (WIII). Unexpectedly, the pbpA or pbpB gene augmentation was conducive to the utilization of sterols. The pbpB augmentation strain WIII-pbpB was further investigated for its better performance. Compared to WIII, the morphology of WIII-pbpB was markedly changed from oval to spindle, indicating alterations of the cell wall. Biochemical analysis indicated that the altered cell wall properties of WIII-pbpB might contribute to the positive effect on sterol utilization. The productivity of 4-HBC was enhanced by 28% in the WIII-pbpB strain compared to that of WIII. These results demonstrated that the modification of peptidoglycan synthesis can improve the conversion of sterols to steroid synthons in mycobacteria.

Enhancement of 9α-Hydroxy-4-androstene-3,17-dione Production from Soybean Phytosterols by Deficiency of a Regulated Intramembrane Proteolysis Metalloprotease in Mycobacterium neoaurum.

Modification of the sterol catabolism pathway in mycobacteria may result in the accumulation of some valuable steroid pharmaceutical intermediates, such as 9α-hydroxy-4-androstene-3,17-dione (9-OHAD). In previous work, sigma factor D (SigD) was identified as a negative factor of the 9-OHAD production in Mycobacterium neoaurum. Here, the deficiency of rip1 putatively coding for a regulated intramembrane proteolysis metalloprotease (Rip1), which could cleave the negative regulator of SigD (anti-SigD), enhanced the transcription of some key genes (choM1, kshA, and hsd4A) in the sterol catabolic pathway. Furthermore, the deletion of rip1 increased the consumption of phytosterols by 37.8% after 96 h of growth in M. neoaurum. The production of 9-OHAD in the engineered M. neoaurumΔkstD1ΔkstD2ΔkstD3Δrip1 (MnΔk123Δrip1) strain was ultimately increased by 27.3% compared to that in its parental strain M. neoaurumΔkstD1ΔkstD2ΔkstD3 (MnΔk123). This study further confirms the important role of SigD-related factors in the catabolism of sterols.

Improving the production of 22-hydroxy-23,24-bisnorchol-4-ene-3-one from sterols in Mycobacterium neoaurum by increasing cell permeability and modifying multiple genes.

BACKGROUND: The strategy of modifying the sterol catabolism pathway in mycobacteria has been adopted to produce steroidal pharmaceutical intermediates, such as 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC), which is used to synthesize various steroids in the industry. However, the productivity is not desirable due to some inherent problems, including the unsatisfactory uptake rate and the low metabolic efficiency of sterols. The compact cell envelope of mycobacteria is a main barrier for the uptake of sterols. In this study, a combined strategy of improving the cell envelope permeability as well as the intracellular sterol metabolism efficiency was investigated to increase the productivity of 4-HBC. RESULTS: MmpL3, encoding a transmembrane transporter of trehalose monomycolate, is an important gene influencing the assembly of mycobacterial cell envelope. The disruption of mmpL3 in Mycobacterium neoaurum ATCC 25795 significantly enhanced the cell permeability by 23.4% and the consumption capacity of sterols by 15.6%. Therefore, the inactivation of mmpL3 was performed in a 4-HBC-producing strain derived from the wild type M. neoaurum and the 4-HBC production in the engineered strain was increased by 24.7%. Subsequently, to enhance the metabolic efficiency of sterols, four key genes, choM1, choM2, cyp125, and fadA5, involved in the sterol conversion pathway were individually overexpressed in the engineered mmpL3-deficient strain. The production of 4-HBC displayed the increases of 18.5, 8.9, 14.5, and 12.1%, respectively. Then, the more efficient genes (choM1, cyp125, and fadA5) were co-overexpressed in the engineered mmpL3-deficient strain, and the productivity of 4-HBC was ultimately increased by 20.3% (0.0633 g/L/h, 7.59 g/L 4-HBC from 20 g/L phytosterol) compared with its original productivity (0.0526 g/L/h, 6.31 g/L 4-HBC from 20 g/L phytosterol) in an industrial resting cell bio-transformation system. CONCLUSIONS: Increasing cell permeability combined with the co-overexpression of the key genes (cyp125, choM1, and fadA5) involved in the conversion pathway of sterol to 4-HBC was effective to enhance the productivity of 4-HBC. The strategy might also be useful for the conversion of sterol to other steroidal intermediates by mycobacteria.